Inhibitory polynucleotide compositions and methods for treating cancer

ABSTRACT

Compositions and methods for treating diseases, such as cancers. The compositions are effective to silence, down-regulate or suppress the expression of a validated target gene by stimulating the process of RNA interference of gene expression, thus inhibiting tumor growth. The invention also provides methods for treating diseases, such as cancers, by inactivation of a validated target gene product, using neutralizing antibody or small molecule drug, to inhibit tumor growth. More particularly, the compositions and methods are directed toward a cancer or a precancerous growth in a mammal, associated with pathological expression of a certain target genes identified herein. The compositions inhibit expression of the target gene when introduced into a tissue of the mammal. The methods include administering the compositions of the invention to a subject in need thereof in an amount effective to inhibit expression of a target gene in a cancerous tissue or organ.

FIELD OF THE INVENTION

The present invention relates generally to polynucleotides useful toinduce RNA interference as a modality in the treatment of cancer. Moreparticularly, the invention relates to target oligonucleotide sequencesdirected toward certain genes implicated in the proliferation and/ormetastasis of precancerous cells, cancer cells or tumor cells.

BACKGROUND OF THE INVENTION

Cancer or pre-cancerous growth generally refers to malignant tumors,rather than benign tumors. Malignant tumors grow faster than benigntumors, and they penetrate and destroy local tissues. Some malignanttumors may spread by metastasis throughout the body via blood or thelymphatic system. The unpredictable and uncontrolled growth makesmalignant cancers dangerous, and fatal in many cases.

Therapeutic treatment of malignant cancer is most effective at the earlystage of cancer development. It is thus exceedingly important toidentify and validate a therapeutic target in early tumor formation andto determine potent tumor growth or gene expression suppression elementsor agents associated therewith.

RNA interference (RNAi) is a post-transcriptional process where in whichdouble-stranded RNA (dsRNA) inhibits gene expression in a sequencespecific fashion. The RNAi process occurs in at least two steps: infirst step, the longer dsRNA is cleaved by an endogenous ribonucleaseDicer into shorter dsRNAs, termed “small interfering RNAs” or siRNAsthat are typically less than 100-, 50-, 30-, 23-, or 21-nucleotides inlength. In the second step, these siRNAs are incorporated into amulticomponent-ribonuclease called RNA-induced-silencing-complex (RISC;Hammond, S. M., et al., Nature (2000) 404:293-296). One strand of siRNAremains associated with RISC, and guides the complex towards a cognateRNA that has sequence complementary to the guider ss-siRNA in RISC. ThissiRNA-directed endonuclease digests the RNA, thereby inactivating it.This RNAi effect can be achieved by introducing either longer dsRNA orshorter siRNA to the target sequence within cells. It is alsodemonstrated that RNAi effect can be achieved by introducing plasmidsthat generate dsRNA complementary to target gene. See WO 99/32619 (Fireet al.); WO 99/53050 (Waterhouse et al.); WO 99/61631 (Heifetz et al.);Yang, D., et al., Curr. Biol. (2000)10:1191-1200), WO 00/44895 (Limmer);and DE 101 00 586.5 (Kreutzer et al.) for disclosures concerning RNAi ina wide range of organisms.

RNAi has been successfully used in gene function determination inDrosophila (Kennerdell et al. (2000) Nature Biotech 18: 896-898; Worbyet al. (2001) Sci STKE Aug. 14, 2001(95):PL1; Schmid et al. (2002)Trends Neurosci 25(2):71-74; Hammond et al. (2000). Nature, 404:293-298), C. elegans (Tabara et al (1998) Science 282: 430-431; Kamathet al. (2000) Genome Biology 2: 2.1-2.10; Grishok et al. (2000) Science287: 2494-2497), and Zebrafish (Kennerdell et al. (2000) Nature Biotech18: 896-898). There are numerous reports on RNAi effects in non-humanmammalian and human cell cultures (Manche et al. (1992). Mol. Cell.Biol. 12:5238-5248; Minks et al. (1979). J. Biol. Chem. 254:10180-10183;Yang et al. (2001) Mol. Cell. Biol. 21(22):7807-7816; Paddison et al.(2002). Proc. Natl. Acad. Sci. USA 99(3):1443-1448; Elbashir et al.(2001) Genes Dev 15(2):188-200; Elbashir et al. (2001) Nature 411:494-498; Caplen et al. (2001) Proc. Natl. Acad. Sci. USA 98: 9746-9747;Holen et al. (2002) Nucleic Acids Research 30(8):1757-1766; Elbashir etal. (2001) EMBO J 20: 6877-6888; Jarvis et al. (2001) TechNotes 8(5):3-5; Brown et al. (2002) TechNotes 9(1): 3-5; Brummelkamp et al. (2002)Science 296:550-553; Lee et al. (2002) Nature Biotechnol. 20:500-505;Miyagishi et al. (2002) Nature Biotechnol. 20:497-500; Paddison et al.(2002) Genes & Dev. 16:948-958; Paul et al. (2002) Nature Biotechnol,20:505-508; Sui et al. (2002) Proc. Natl. Acad. Sci. USA99(6):5515-5520; Yu et al. (2002) Proc. Natl. Acad. Sci. USA99(9):6047-6052).

SUMMARY OF THE INVENTION

The present invention provides compositions and methods for treatingdiseases, such as cancers. The compositions are effective to silence,down-regulate or suppress the expression of a validated target gene bystimulating the process of RNA interference of gene expression. Thecompositions and methods thereby inhibit tumor growth. The inventionalso provides methods for treating diseases, such as cancers, byinactivation of a validated target gene product, using neutralizingantibody or small molecule drug, to inhibit tumor growth.

More particularly, the compositions and methods are directed toward acancer or a precancerous growth in a mammal, associated withpathological expression of a target gene chosen from among an ICT-1053gene, or an ICT-1052 gene, or an ICT-1027 gene, or an ICT-1051 gene, oran ICT-1054 gene, or an ICT-1020 gene, or an ICT-1021 gene, or anICT-1022 gene (a “Target Gene” or “Target Genes” herein). Thecompositions inhibit expression of the target gene when introduced intoa tissue of the mammal. The methods include administering thecompositions of the invention to a subject in need thereof in an amounteffective to inhibit expression of a target gene in a cancerous tissueor organ.

In a first aspect the invention provides an isolated targetingpolynucleotide whose length is 200 or fewer nucleotides. Thispolynucleotide includes a first nucleotide sequence that targets aTarget Gene or a complement thereto. The first nucleotide sequence orits complement is any number of nucleotides from 15 to 30 in length, andin several embodiments the length is 21 to 25 nucleotides.

In another aspect the polynucleotide of the invention described in thepreceding paragraph further includes a second nucleotide sequenceseparated from the first nucleotide sequence by a loop sequence; thesecond nucleotide sequence

-   -   a) has substantially the same length as the first nucleotide        sequence, and    -   b) is substantially complementary to the first nucleotide        sequence, such that the polynucleotide forms a hairpin structure        under conditions suitable for hybridization of the first and        second nucleotide sequences.        In many embodiments of the linear polynucleotide hairpin        polynucleotide described in the preceding paragraphs, the first        nucleotide sequence consists of    -   a) a sequence that targets a sequence chosen from SEQ ID        NOS:7-76, 81-84, and 89-242 (a “Target Sequence” herein);    -   b) an extended sequence longer than, and containing, the        targeting sequence given in item a), wherein the extended        sequence targets a Target Gene, and the targeting sequence        targets a Target Sequence;    -   c) a fragment of a sequence that targets a Target Sequence at        least 15 nucleotides long, and shorter than the chosen Target        Sequence;    -   d) a targeting sequence wherein up to 5 nucleotides differ from        a chosen Target Sequence; or    -   e) a complement of a sequence given in a)-d).

In common embodiments the linear polynucleotide described hereinconsists of a Target Sequence, and optionally includes a dinucleotideoverhang bound to the 3′ of the chosen sequence. In related commonembodiments the hairpin polynucleotide described herein consists of afirst chosen Target nucleotide Sequence, a loop sequence and the secondnucleotide sequence substantially complementary to the Target Sequence.

In further embodiments the polynucleotide is a DNA, or an RNA, or thepolynucleotide includes both deoxyribonucleotides and ribonucleotides.

In an additional aspect the invention provides a double strandedpolynucleotide containing a first targeting linear polynucleotide stranddescribed herein and a second polynucleotide strand including a second,nucleotide sequence that is substantially complementary to at least thefirst nucleotide sequence of the first polynucleotide strand and ishybridized thereto.

In still a further aspect the invention provides a combination ormixture of polynucleotides that includes a plurality of targeting linearpolynucleotides, double stranded polynucleotides and/or hairpinpolynucleotides described herein wherein each polynucleotide targets adifferent chosen Target Sequence in one or more chosen Target Genes.

In yet an additional aspect the invention provides a vector containingthe targeting linear polynucleotide or the targeting hairpinpolynucleotide described herein. In common embodiments the vector is aplasmid, a cosmid, a recombinant virus, a retroviral vector, anadenoviral vector, a transposon, or a minichromosome.

In further common embodiments of the vector a control element isoperatively linked with the targeting polynucleotide effective topromote expression thereof. Additional aspects provide a celltransfected with one or more linear polynucleotides described herein ora cell transfected with one or more hairpin polynucleotides describedherein, or a cell transfected with a combination of the saidpolynucleotides.

In still a further aspect the invention provides a pharmaceuticalcomposition containing one or more linear polynucleotides or hairpinpolynucleotides described herein, or a mixture thereof, wherein eachpolynucleotide targets a different Target Sequence in a Target Gene, orany two or more thereof, and a pharmaceutically acceptable carrier.

In yet an additional aspect the invention provides a method ofsynthesizing a polynucleotide having a sequence that targets a TargetGene described herein. The methods includes the steps of

-   -   a) providing a nucleotide reagent including a live reactive end        and corresponding to the nucleotide at a first end of the        sequence,    -   b) adding a further nucleotide reagent including a live reactive        end and corresponding to a successive position of the sequence        to react with the live reactive end from the preceding step and        increase the length of the growing polynucleotide sequence by        one nucleotide, and removing undesired products and excess        reagent, and    -   c) repeating step b) until the nucleotide reagent corresponding        to the nucleotide at a second end of the sequence has been        added;    -   thereby providing the completed polynucleotide.

In still a further aspect the invention provides a method of inhibitingthe growth of a cancer cell that includes contacting the cell with acomposition containing one or more targeting linear polynucleotides ortargeting hairpin polynucleotides described herein or a mixture thereoftinder conditions promoting incorporation of the one or morepolynucleotides within the cell.

In an additional aspect the invention provides a method of promotingapoptosis in a cancer cell that includes contacting the cell with acomposition containing one or more targeting linear polynucleotides ortargeting hairpin polynucleotides described herein or a mixture thereofunder conditions promoting incorporation of the one or morepolynucleotides within the cell.

In yet another aspect, the invention provides methods for inhibitingcancer or precancerous growth in a mammalian tissue, wherein the methodincludes contacting the tissue with an inhibitory targetingpolynucleotide of the invention that interacts with DNA or RNA thatcontains one or more Target Genes. The targeting polynucleotide inhibitsexpression of the one or more Target Genes in cells of the tissue. Inseveral embodiments of this method the tissue is a breast tissue, colontissue, a prostate tissue, a skin tissue, a bone tissue, a parotid glandtissue, a pancreatic tissue, a kidney tissue, a uterine cervix tissue, alymph node tissue, or an ovarian tissue. Furthermore the inhibitorytargeting polynucleotide is a nucleic acid molecule, a decoy molecule, adecoy DNA, a double stranded DNA, a single-stranded DNA, a complexedDNA, an encapsulated DNA, a viral DNA, a plasmid DNA, a naked RNA, anencapsulated RNA, a viral RNA, a double stranded RNA, a molecule, orcombinations thereof.

In yet a further aspect the invention provides a use of a targetinglinear polynucleotide or a targeting hairpin polynucleotide describedherein, or of a mixture of two or more of them, wherein eachpolynucleotide targets a Target Gene, in the manufacture of apharmaceutical composition effective to treat a cancer or a precancerousgrowth in a subject. In several embodiments of the use the cancer or thegrowth is found in a tissue chosen from breast tissue, colon tissue,prostate tissue, skin tissue, bone tissue, parotid gland tissue,pancreatic tissue, thyroid tissue, kidney tissue, uterine cervix tissue,lung tissue, lymph node tissue, hematopoietic tissue of bone marrow, orovarian tissue. In additional common embodiments of the use the firstnucleotide sequence in each polynucleotide consists of

-   -   a) a sequence that targets a sequence chosen from SEQ ID        NOS:7-76, 81-84, and 89-242 (a “Target Sequence” herein);    -   b) an extended sequence longer than, and containing, the        targeting sequence given in item a), wherein the extended        sequence targets a Target Gene, and the targeting sequence        targets a Target Sequence;    -   c) a fragment of a sequence that targets a Target Sequence at        least 15 nucleotides long, and shorter than the chosen Target        Sequence;    -   d) a targeting sequence wherein up to 5 nucleotides differ from        a chosen Target Sequence; or    -   e) a complement of a sequence given in a)-d).        In still further embodiments of the use the subject is a human.

In still a further aspect, the invention provides the use one or moreantibodies directed against a product polypeptide of a Target Gene inthe manufacture of a pharmaceutical composition effective to treat acancer, a tumor or a precancerous growth in a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Schematic representation of various embodiments of thepolynucleotides of the invention. Panel A, embodiments of a linearpolynucleotide. The length is 200 nucleotides or less, and 15nucleotides or greater. In b), a specified targeting sequence iscontained within a larger targeting sequence. In d) the darker verticalbars diagrammatically represent substituted nucleotides. Panel B, anembodiment of a hairpin polynucleotide of overall length 200 nucleotidesor less.

FIG. 2. Representation of the change in tumor size of MDA-MB-435xenografts with time in response to transfection with ICT-1053 (PCDP10)siRNA, or with control siRNAs. Data are presented as Mean+/−SE.

FIG. 3. Representation of the change in tumor size of MDA-MB-435xenografts with time in response to transfection with ICT-1052 (cMet)siRNA, or with control siRNAs. Data are presented as Mean+/−SE.

FIG. 4. Representation of the change in tumor size of A549 xenograftswith time in response to transfection with ICT-1052 (cMet) siRNA, orwith a control siRNA. Data are presented as Mean+/−SE.

FIG. 5. Representation of the inhibition of proliferation of MDA-MB-435cells in culture when treated with ICT-1052 siRNA or ICT-1053 siRNA, ora control siRNA. Data are presented as mean values.

FIG. 6. Representation of the inhibition of proliferation of HCT116human colon carcinoma cells in culture when treated with ICT-1052 siRNAor ICT-1053 siRNA, or a control siRNA. Data are presented as mean+/−SE.

FIG. 7. Representation of the inhibition of proliferation of A549 humanlung carcinoma cells in culture when treated with ICT-1052 siRNA or acontrol siRNA. Data are presented as mean+/−SE.

FIG. 8. Representation of the change in tumor size of MDA-MB-435xenografts with time in response to transfection with ICT-1027 (GRB2 BP)siRNA or a control siRNA. Data were presented as mean+/−SE.

FIG. 9. Representation of the induction of apoptosis in MDA-MB-435 cellsin response to treatment with ICT-1027 siRNA, or control siRNA. Data arepresented as mean+/−SE.

FIG. 10. Representation of the change in tumor size of MDA-MB-435xenografts with time in response to transfection with ICT-1051 (A-Raf)siRNA or a control siRNA. Data were presented as mean+/−SE.

FIG. 11. Representation of the change in tumor size of MDA-MB-435xenografts with time in response to transfection with ICT-1054 (PCDP6)siRNA or a control siRNA. Data were presented as mean+/−SE.

FIG. 12. Representation of the change in tumor size of MDA-MB-435xenografts with time in response to transfection with ICT-1020 (Dicer)siRNA or a control siRNA. Data were presented as mean+/−SE.

FIG. 13. Representation of the change in tumor size of MDA-MB-435xenografts with time in response to transfection with ICT-1021 (MD2protein) siRNA or a control siRNA. Data were presented as mean+/−SE.

FIG. 14. Representation of the change in tumor size of MDA-MB-435xenografts with time in response to transfection with ICT-1022 (GAGE-2)siRNA or a control siRNA. Data were presented as mean+/−SE.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent application publications, and patent applicationsidentified herein are incorporated by reference in their entireties, asif appearing herein verbatim. All technical publications identifiedherein are also incorporated by reference.

In the present description, the articles “a”, “an”, and “the” relateequivalently to a meaning as singular or as plural. The particular sensefor these articles is apparent from the context in which they are used.

As used herein the term “tumor” refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all precancerous andcancerous cells and tissues.

As used herein the term “precancerous” refers to cells or tissues havingcharacteristics relating to changes that may lead to malignancy orcancer.

As used herein the term “cancer” refers to cells or tissues possessingcharacteristics such as uncontrolled proliferation, loss of specializedfunctions, immortality, significant metastatic potential, significantincrease in anti-apoptotic activity, rapid growth and proliferationrate, and certain characteristic morphological and cellular markers. Insome circumstances, cancer cells will be in the form of a tumor; suchcells may exist locally within an animal, and in other circumstancesthey may circulate in the blood stream as independent cells, forexample, leukemic cells.

As used herein the term “target” sequence and similar terms and phrasesrelate to a nucleotide sequence that occurs in a nucleic acid of acancer cell against which a polynucleotide of the invention is directed.A “target gene” refers to an expressed gene wherein modulation of thelevel of gene expression or of gene product activity prevents and/orameliorates disease progression. In particular, target genes in thepresent invention include endogenous genes and their variants, asdescribed herein.

A targeting polynucleotide targets a cancer cell nucleic acid sequenceeither a) by including a sequence whose complement is homologous oridentical to a particular subsequence (termed a target sequence)contained within the genome of the pathogen, or b) by including asequence that is itself homologous or identical to the target sequence.A targeting polynucleotide that is effective within a cell is a doublestranded molecule comprised of one of each the strands specified in a)and b). It is believed that any double stranded targeting polynucleotideso targeting a cancer cell nucleic acid sequence has the ability tohybridize with the target sequence according to the RNA interferencephenomenon, thereby initiating RNA interference.

A target gene in a subject may have a sequence that is identical to awild type sequence identified, for example, in various GenBank accessionentries, and in entries in similar databases; typically such databasesare accessible to the public. An interfering RNA to be used to suppressexpression of a target gene may, however, not, be perfectlycomplementary to its target, or the target may differ from a sequenceconsidered to be a wild type sequence given by an existing GenBankaccession number. For example, a target gene may include one or moresingle polynucleotide polymorphisms, and thus differ slightly from thesequence in a GenBank accession number. In addition a target gene mayproduce an mRNA that is the product of alternative splicing of exons,resulting in a mature mRNA that has fewer exons than the chromosomalgene. Such an alternatively spliced mRNA can also be a target of an RNAispecies directed against the wild type gene. In the present disclosureall such eventualities are encompassed within the notion of a targetgene, and any RNAi species developed to target the wild type sequencepotentially targets such altered or modified transcripts and is includedwithin the notion of a targeting sequence.

In general, a “gene” is a region in the genome that is capable of beingtranscribed to an RNA that either has a regulatory function, a catalyticfunction, and/or encodes a protein. A eukaryotic gene typically hasintrons and exons, which may organize to produce different RNA splicevariants that encode alternative versions of a mature protein. Theskilled artisan will appreciate that the present invention encompassesall endogenous genes that may be found, including splice variants,allelic variants and transcripts that occur because of alternativepromoter sites or alternative polyadenylation sites. The endogenousgene, as described herein, also can be a mutated endogenous gene,wherein the mutation can be in the coding or regulatory regions.

“Antisense RNA”: In eukaryotes, RNA polymerase catalyzes thetranscription of a structural gene to produce mRNA. A DNA molecule canbe designed to contain an RNA polymerase template in which the RNAtranscript has a sequence that is complementary to that of a preferredmRNA. The RNA transcript is termed an “antisense RNA.” Antisense RNAmolecules can inhibit mRNA expression (for example, Rylova et al.,Cancer Res, 62(3):801-8, 2002; Shim et al., Int. J. Cancer, 94(1):6-15,2001).

“Antisense DNA” or “DNA decoy” or “decoy molecule”: With respect to afirst nucleic acid molecule, a second DNA molecule or a second chimericnucleic acid molecule that is created with a sequence, which is acomplementary sequence or homologous to the complementary sequence ofthe first molecule or portions thereof, is referred to as the antisenseDNA or DNA decoy or decoy molecule of the first molecule. The term“decoy molecule” also includes a nucleic molecule, which may be singleor double stranded, that comprises DNA or PNA (peptide nucleic acid)(Mischiati et al., Int. J. Mol. Med., 9(6):633-9, 2002), and thatcontains a sequence of a protein binding site, preferably a binding sitefor a regulatory protein and more preferably a binding site for atranscription factor. Applications of antisense nucleic acid molecules,including antisense DNA and decoy DNA molecules are known in the art,for example, Morishita et al., Ann. N Y Acad. Sci., 947:294-301, 2001;Andratschke et al., Anticancer Res, 21:(5)3541-3550, 2001.

“Stabilized RNA”: A stabilized RNAi, siRNA or a shRNA as describedherein, is protected against degradation by exonucleases, includingRNase, for example, using a nucleotide analogue that is modified at the3′ position of the ribose sugar (for example, by including a substitutedor unsubstituted alkyl, alkoxy, alkenyl, alkenyloxy, alkynyl oralkynyloxy group as defined above), or modified elsewhere in itsstructure to achieve protection. The RNAi, siRNA or a shRNA also can bestabilized against degradation at the 3′ end by exonucleases byincluding a 3′-3′-linked dinucleotide structure (Ortigao et al.,Antisense Research and Development 2:129-146 (1992)) and/or two modifiedphospho bonds, such as two phosphorothioate bonds.

“Encapsulated nucleic acids”, including encapsulated DNA or encapsulatedRNA, refer to nucleic acid molecules in microsphere or microparticle andcoated with materials that are relatively non-immunogenic and subject toselective enzymatic degradation, for example, synthesized microspheresor microparticles by the complex coacervation of materials, for example,gelatin and chondroitin sulfate (see, for example, U.S. Pat. No.6,410,517). Encapsulated nucleic acids in a microsphere or amicroparticle are encapsulated in such a way that it retains its abilityto induce expression of its coding sequence (see, for example, U.S. Pat.No. 6,406,719).

“Inhibitors” refers to molecules that inhibit and/or block an identifiedfunction. Any molecule having potential to inhibit and/or block anidentified function can be a “test molecule,” as described herein. Forexample, referring to oncogenic function or anti-apoptotic activity of aTarget Gene, such molecules may be identified using in vitro and in vivoassays of the particular Target Gene. Inhibitors are compounds thatpartially or totally block Target Gene activity, decrease, prevent, ordelay their activation, or desensitize its cellular response. This maybe accomplished by binding to Target Gene products, i.e. proteins,directly or via other intermediate molecules. An antagonist or anantibody, e.g. monoclonal or polyclonal antibody, that blocks geneproduct activity of a Target Gene, including inhibition of oncogenicfunction or anti-apoptotic activity of a Target Gene, is considered tobe such an inhibitor. Inhibitors according to the instant invention is:a siRNA, an RNAi, a shRNA, an antisense RNA, an antisense DNA, a decoymolecule, a decoy DNA, a double stranded DNA, a single-stranded DNA, acomplexed DNA, an encapsulated DNA, a viral DNA, a plasmid DNA, a nakedRNA, an encapsulated RNA, a viral RNA, a double stranded RNA, a moleculecapable of generating RNA interference, or combinations thereof. Thegroup of inhibitors of this invention also includes genetically modifiedversions of Target Genes, for example, versions with altered activity.The group thus is inclusive of the naturally occurring protein as wellas synthetic ligands, antagonists, agonists, antibodies, small chemicalmolecules and the like.

“Assays for inhibitors” refer to experimental procedures including, forexample, expressing Target Genes in vitro, in cells, applying putativeinhibitor compounds, and then determining the functional effects onTarget Gene activity or transcription. Samples that contain or aresuspected of containing a Target Gene are treated with a potentialinhibitor. These inhibitors include nucleic acid based molecules, suchas siRNA, antisense, double-stranded RNA and DNA, or double-strandedRNA/DNA, ribozyme and triplex, etc.; and protein based molecules, suchas peptides, synthetic ligands, truncated partial proteins, solublereceptors, monoclonal antibody, polyclonal antibody, intrabody andsingle chain antibody, etc.; as well as small chemical molecules atvarious forms. The extent of inhibition or change is examined bycomparing the activity measurement from the samples of interest tocontrol samples. A threshold level is established to assess inhibition.For example, inhibition of a Target Gene product polypeptide isconsidered achieved when the Target Gene activity value relative to asuitable control is 80% or lower.

As used herein, a first sequence or subsequence is “identical”, or has“100% identity”, or is described by a term or phrase conveying thenotion of 100% identity, to a second sequence or subsequence when thefirst sequence or subsequence has the same base as the second sequenceor subsequence at every position of the sequence or subsequence. Indetermining identity, any T (thymidine) or any derivative thereof, or aU (uridine) or any derivative thereof, are equivalent to each other, andthus identical. No gaps are permitted for a first and second sequence tobe identical.

A sequence of a targeting polynucleotide, or its complement, may becompletely identical to the target sequence, or it may includemismatched bases at particular positions in the sequence. Incorporationof mismatches is described fully herein. Without wishing to be bound bytheory, it is believed that incorporation of mismatches provides anintended degree of stability of hybridization under physiologicalconditions to optimize the RNA interference phenomenon for theparticular target sequence in question. The extent of identitydetermines the percent of the positions in the two sequences whose basesare identical to each other. The “percentage of sequence identity” iscalculated as shown

${\% \mspace{14mu} {Identity}} = {\frac{{Number}\mspace{14mu} {of}\mspace{14mu} {identical}\mspace{14mu} {bases}}{{Total}\mspace{14mu} {number}\mspace{14mu} {of}\mspace{14mu} {bases}} \times 100}$

Sequences that are less than 100% identical to each other are “similar”or “homologous” to each other; the degree of homology or the percentsimilarity are synonymous terms relating to the percent of identitybetween two sequences or subsequences. For example, two sequencesdisplaying at least 60% identity, or preferably at least 65% identity,or preferably at least 70% identity, or preferably at least 75%identity, or preferably at least 80% identity, or more preferably atleast 85% identity, or more preferably at least 90% identity, or stillmore preferably at least 95% identity, to each other are “similar” or“homologous” to each other. Alternatively; with reference to theoligonucleotide sequence of an siRNA molecule, two sequences that differby 5 or fewer bases, or by 4 or fewer bases, or by 3 or fewer bases, orby two or fewer bases, or by one base, are termed “similar” or“homologous” to each other.

“Identity” and “similarity” can additionally be readily calculated byknown methods, including but not limited to those described inComputational Molecular Biology, Lesk. A. M., ed., Oxford UniversityPress, New York, 1988; Biocomputing: Informatics and Genome Projects,Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis ofSequence Data, Part I. Griffin, A. M., and Griffin, H. G., eds. HumanaPress, New Jersey, 1994; Sequence Analysis in Molecular Biology, vonHeinje, G., Academic Press, 1987; and Sequence Analysis Primer,Gribskov, M. and Devereux, J., eds., M Stockton Press. New York, 1991;and Carillo, H., and Lipman, D., SIAM J. Applied Math. (1988) 48: 1073.Methods of alignment of sequences for comparison are well-known in theart. Optimal alignment of sequences for comparison may be conducted bythe local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482, 1981; by the homology alignment algorithm of Needleman and Wunsch,J. Mol. Biol., 48: 443, 1970; by the search for similarity method ofPearson and Lipman, Proc. Natl. Acad. Sci. USA, 8: 2444, 1988; bycomputerized implementations of these algorithms, including, but notlimited to: CLUSTAL in the PC/Gene program by Intelligenetics, MountainView, Calif., GAP, BESTFIT, BLAST, FASTA, and TFASTA in the WisconsinGenetics Software Package, Genetics Computer Group (GCG), 7 Science Dr.,Madison, Wis., USA; the CLUSTAL program is well described by Higgins andSharp, Gene, 73: 237-244, 1988; Corpet, et al., Nucleic Acids Research,16:881-90, 1988; Huang, et al., Computer Applications in theBiosciences, 8:1-6, 1992; and Pearson, et al., Methods in MolecularBiology, 24:7-331, 1994. The BLAST family of programs which can be usedfor database similarity searches includes: BLASTN for nucleotide querysequences against nucleotide database sequences; BLASTX for nucleotidequery sequences against protein database sequences; BLASTP for proteinquery sequences against protein database sequences; TBLASTN for proteinquery sequences against nucleotide database sequences; and TBLASTX fornucleotide query sequences against nucleotide database sequences. See,Current Protocols in Molecular Biology, Chapter 19, Ausubel, et al.,Eds., Greene Publishing and Wiley-Interscience, New York, 1995.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using, the BLAST 2.0 suite ofprograms, or their successors, using default parameters. Altschul etal., Nucleic Acids Res, 2:3389-3402, 1997. It is to be understood thatdefault settings of these parameters can be readily changed as needed inthe future.

The term “substantial identity” or “homologous” in their variousgrammatical forms means that a polynucleotide comprises a sequence thathas a desired identity, for example, at least 60% identity, preferablyat least 70% sequence identity, more preferably at least 80%, still morepreferably at least 90% and even more preferably at least 95%, comparedto a reference sequence using one of the alignment programs described.

As used herein, the term “isolated”, and similar words, when used todescribe a nucleic acid, a polynucleotide, or an oligonucleotide relateto the composition being removed from its natural or original state.Thus, if it occurs in nature, it has been removed from its originalenvironment. If it has been prepared synthetically, it has been removedfrom an original product mixture resulting from the synthesis. Forexample, a naturally occurring polynucleotide naturally present in aliving organism in its natural state is not “isolated,” but the samepolynucleotide separated from at least one material with which itcoexists in its natural state is “isolated”, as the term is employedherein. Generally, removal of at least one coexisting materialconstitutes “isolating” a nucleic acid, a polynucleotide, anoligonucleotide. In many cases several, many, or most coexistingmaterials may be removed to isolate the nucleic acid, polynucleotide, oroligonucleotide. A nucleic acid, a polynucleotide, or an oligonucleotidethat is the product of an in vitro synthetic process or a chemicalsynthetic process is essentially isolated as the result of the syntheticprocess. In important embodiments such synthetic products are treated toremove reagents and precursors used, and side products produced, by theprocess.

Polynucleotides incorporated into a composition, such as a formulation,a transfecting composition, a pharmaceutical composition, orcompositions or solutions for chemical or enzymatic reactions, which arenot naturally occurring compositions, remain isolated polynucleotides orpolypeptides within the meaning of that term as it is employed herein.

As used herein, a “nucleic acid” or “polynucleotide”, and similar termsand phrases, relate to polymers composed of naturally occurringnucleotides as well as to polymers composed of synthetic or modifiednucleotides. Thus, as used herein, a polynucleotide that is a RNA, or apolynucleotide that is a DNA, or a polynucleotide that contains bothdeoxyribonucleotides and ribonucleotides, may include naturallyoccurring moieties such as the naturally occurring bases and ribose ordeoxyribose rings, or they may be composed of synthetic or modifiedmoieties such as those described below. A polynucleotide employed in theinvention may be single stranded or it may be a base paired doublestranded structure, or even a triple stranded base paired structure.

Nucleic acids and polynucleotides may be 20 or more nucleotides inlength, or 30 or more nucleotides in length, or 50 or more nucleotidesin length, or 100 or more, or 1000 or more, or tens of thousands ormore, or, hundreds of thousands or more, in length. An siRNA may be apolynucleotide as defined herein. As used herein, “oligonucleotides” andsimilar terms based on this relate to short polymers composed ofnaturally occurring nucleotides as well as to polymers composed ofsynthetic or modified nucleotides, as described in the immediatelypreceding paragraph. Oligonucleotides may be 10 or more nucleotides inlength, or 15, or 16, or 17, or 18, or 19, or 20 or more nucleotides inlength, or 21, or 22, or 23, or 24 or more nucleotides in length, or 25,or 26, or 27, or 28 or 29, or 30 or more nucleotides in length, 35 ormore, 40 or more, 45 or more, up to about 50, nucleotides in length. Anoligonucleotide sequence employed as a targeting sequence in an siRNAmay have any number of nucleotides between 15 and 30 nucleotides. Inmany embodiments an siRNA may have any number of nucleotides between 21and 25 nucleotides. Oligonucleotides may be chemically synthesized andmay be used as siRNAs, PCR primers, or probes.

It is understood that, because of the overlap in size ranges provided inthe preceding paragraph, the terms “polynucleotide” and“oligonucleotide” may be used synonymously herein to refer to an siRNAof the invention.

As used herein “nucleotide sequence”, “oligonucleotide sequence” or“polynucleotide sequence”, and similar terms, relate interchangeablyboth to the sequence of bases that an oligonucleotide or polynucleotidehas, as well as to the oligonucleotide or polynucleotide structurepossessing the sequence. A nucleotide sequence or a polynucleotidesequence furthermore relates to any natural or synthetic polynucleotideor oligonucleotide in which the sequence of bases is defined bydescription or recitation of a particular sequence of lettersdesignating bases as conventionally employed in the field.

A “nucleoside” is conventionally understood by workers of skill infields such as biochemistry, molecular biology, genomics, and similarfields related to the field of the invention as comprising amonosaccharide linked in glycosidic linkage to a purine or pyrimidinebase; and a “nucleotide” comprises a nucleoside with at least onephosphate group appended, typically at a 3′ or a 5′ position (forpentoses) of the saccharide, but may be at other positions of thesaccharide. Nucleotide residues occupy sequential positions in anoligonucleotide or a polynucleotide. A modification or derivative of anucleotide may occur at any sequential position in an oligonucleotide ora polynucleotide. All modified or derivatized oligonucleotides andpolynucleotides are encompassed within the invention and fall within thescope of the claims. Modifications or derivatives can occur in thephosphate group, the monosaccharide or the base.

By way of nonlimiting examples, the following descriptions providecertain modified or derivatized nucleotides, all of which are within thescope of the polynucleotides of the invention. The monosaccharide may bemodified by being, for example, a pentose or a hexose other than aribose or a deoxyribose. The monosaccharide may also be modified bysubstituting hydroxyl groups with hydro or amino groups, by alkylatingor esterifying additional hydroxyl groups, and so on. Substituents atthe 2′ position, such as 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl,2′-O-allyl, 2′-O-aminoalkyl or 2′-deoxy-2′-fluoro group provide enhancedhybridization properties to an oligonucleotide.

The bases in oligonucleotides and polynucleotides may be “unmodified” or“natural” bases include the purine bases adenine (A) and guanine (G),and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Inaddition they may be bases with modifications or substitutions.Nonlimiting examples of modified bases include other synthetic andnatural bases such as hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine, 5-hydroxymethyl cytosine, xanthine, hypoxanthine,2-aminoadenine, 6-methyl and other alkyl derivatives of adenine andguanine, 2-propyl and other alkyl derivatives of adenine and guanine,2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil,5-halo-cytosine, 5-propy-uracil, 5-propynyl-cytosine and other alkynylderivatives of pyrimidine bases, 6-azo-uracil, 6-azo-cytosine,6-azo-thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino-,8-thiol-, 8-thioalkyl-, 8-hydroxyl- and other 8-substituted adenines andguanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 2-fluoro-adenine, 2-amino-adenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and3-deazaadenine. Further modified bases include tricyclic pyrimidinessuch as phenoxazinecytidine(1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazinecytidine (1-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), G-clamps suchas a substituted phenoxazine cytidine (e.g.9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazolecytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine(H-pyrido[3′, 2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified bases mayalso include those in which the purine or pyrimidine base is replacedwith other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine,2-aminopyridine and 2-pyridone. Further bases include those disclosed inU.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, those disclosed by Englisch et al., AngewandteChemie, International Edition (1991) 30, 613, and those disclosed bySanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain ofthese bases are particularly useful for increasing the binding affinityof the oligomeric compounds of the invention. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2.degree. C.(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Researchand Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and arepresently preferred base substitutions, even more particularly whencombined with 2′-O-methoxyethyl sugar modifications. See U.S. Pat. Nos.6,503,754 and 6,506,735 and references cited therein, incorporatedherein by reference. Modifications further include those disclosed inU.S. Pat. Nos. 5,138,045 and 5,218,105, drawn to polyamine conjugatedoligonucleotides; U.S. Pat. Nos. 5,212,295, 5,521,302, 5,587,361 and5,599,797, drawn to oligonucleotides incorporating chiral phosphoruslinkages including phosphorothioates; U.S. Pat. Nos. 5,378,825,5,541,307, and 5,386,023, drawn to oligonucleotides having modifiedbackbones; U.S. Pat. Nos. 5,457,191 and 5,459,255, drawn to modifiednucleobases; U.S. Pat. No. 5,539,082, drawn to peptide nucleic acids;U.S. Pat. No. 5,554,746, drawn to oligonucleotides having beta-lactambackbones; U.S. Pat. No. 5,571,902, disclosing the synthesis ofoligonucleotides; U.S. Pat. No. 5,578,718, disclosing alkylthionucleosides; U.S. Pat. No. 5,506,351, drawn to 2′-O-alkyl guanosine,2,6-diaminopurine, and related compounds; U.S. Pat. No. 5,587,469, drawnto oligonucleotides having N-2 substituted purines; U.S. Pat. No.5,587,470, drawn to oligonucleotides having 3-deazapurines; U.S. Pat.No. 5,223,168, and U.S. Pat. No. 5,608,046, drawn to conjugated4′-desmethyl nucleoside analogs; U.S. Pat. Nos. 5,602,240, and5,610,289, drawn to backbone-modified oligonucleotide analogs; U.S. Pat.Nos. 6,262,241, and 5,459,255, drawn to, inter alia, methods ofsynthesizing 2′-fluoro-oligonucleotides.

The linkages between nucleotides is commonly the 3′-5′ phosphatelinkage, which may be a natural phosphodiester linkage, aphosphothioester linkage, and still other synthetic linkages.Oligonucleotides containing phosphorothioate backbones have enhancednuclease stability. Examples of modified backbones include,phosphorothioates, chiral phosphorothioates, phosphorodithioates,phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates including 3′-alkylene phosphonates, 5′-alkylenephosphonates and chiral phosphonates, phosphinates, phosphoramidatesincluding 3′-amino phosphoramidate and aminoalkylphosphoramidates,thionophosphoramidates, thionoalkylphosphonates,thionoalkylphosphotriesters, selenophosphates and boranophosphates.Additional linkages include phosphotriester, siloxane, carbonate,carboxymethylester, acetamidate, carbamate, thioether, bridgedphosphoramidate, bridged methylene phosphonate, bridged phosphorothioateand sulfone internucleotide linkages. Other polymeric linkages include2′-5′ linked analogs of these. See U.S. Pat. Nos. 6,503,754 and6,506,735 and references cited therein, incorporated herein byreference.

Any modifications including those exemplified in the above descriptioncan readily be incorporated into, and are comprised within the scope of,the targeting polynucleotides of the invention. Use of any modifiednucleotide is equivalent to use of a naturally occurring nucleotidehaving the same base-pairing properties, as understood by a worker ofskill in the art. All equivalent modified nucleotides fall within thescope of the present invention as disclosed and claimed herein.

As used herein and in the claims, the term “complement”,“complementary”, “complementarity”, and similar words and phrases,relate to two sequences whose bases form complementary base pairs, baseby base, as conventionally understood by workers of skill in fields suchas biochemistry, molecular biology, genomics, and similar fields relatedto the field of the invention. Two single stranded (ss) polynucleotideshaving complementary sequences can hybridize with each other undersuitable buffer and temperature conditions to form a double stranded(ds) polynucleotide. By way of nonlimiting example, if the naturallyoccurring bases are considered, A and (T or U) interact with each other,and G and C interact with each other. Unless otherwise indicated,“complementary” is intended to signify “fully complementary”, namely,that when two polynucleotide strands are aligned with each other, therewill be at least a portion of the strands in which each base in asequence of contiguous bases in one strand is complementary to aninteracting base in a sequence of contiguous bases of the same length onthe opposing strand.

As used herein, “hybridize”, “hybridization” and similar words andphrases relate to a process of forming a nucleic acid, polynucleotide,or oligonucleotide duplex by causing strands with complementarysequences to interact with each other. The interaction occurs by virtueof complementary bases on each of the strands specifically interactingto form a pair. The ability of strands to hybridize to each otherdepends on a variety of conditions, as set forth below. Nucleic acidstrands hybridize with each other when a sufficient number ofcorresponding positions in each strand are occupied by nucleotides thatcan interact with each other. Polynucleotide strands that hybridize toeach other may be fully complementary. Alternatively, two hybridizedpolynucleotides may be “substantially complementary” to each other,indicating that they have a small number of mismatched bases. Bothnaturally occurring bases, and modified bases such as those describedherein, participate in forming complementary base pairs. It isunderstood by workers of skill in the field of the present invention,including by way of nonlimiting example biochemists and molecularbiologists, that the sequences of strands forming a duplex need not be100% complementary to each other to be specifically hybridizable.

As used herein, a “nucleotide overhang” and similar terms and phrasesrelate to an unpaired nucleotide, or nucleotides that extend beyond theduplex structure of a double stranded polynucleotide when a 3′-end ofone strand of the duplex extends beyond the 5′-end of the other strand,or mutatis mutandi. Conversely “blunt” or “blunt end” and similar termsand phrases relate to a duplex having no unpaired nucleotides at an endof the duplex, i.e., no nucleotide overhang.

As used herein, “antisense strand” and similar terms and phrases relateto a strand of a polynucleotide duplex which includes a region that issubstantially complementary to a target sequence. As used herein, theterm “region of complementarity” refers to the region on the antisensestrand that is substantially complementary to a sequence, for example atarget sequence, as defined herein. If a region of complementarity isnot fully complementary to a target sequence, mismatches are commonlytolerated in the terminal regions and, if present, are commonly within6, 5, 4, 3, or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand” and similar terms and phrases as used herein,relate to a strand of a polynucleotide duplex that includes a regionthat is complementary to a region of the antisense strand of a targetsequence. Thus a sense strand has a region that is identical orsubstantially similar to the target sequence.

As used herein “fragment” and similar words and phrases relate toportions of a nucleic acid, polynucleotide or oligonucleotide shorterthan the full sequence of a reference. The sequence of bases in afragment is unaltered from the sequence of the corresponding portion ofthe reference; there are no insertions or deletions in a fragment incomparison with the corresponding portion of the reference. Ascontemplated herein, a fragment of a nucleic acid or polynucleotide,such as an oligonucleotide, is 15 or more bases in length, or 16 ormore, 17 or more, 18 or more, or 19 or more, or 20 or more, or 21 ormore, or 22 or more, or 23 or more, or 24 or more, or 25 or more, or 26or more, or 27 or more, or 28 or more, or 29 or more, or 30 or more, or50 or more, or 75 or more, or 100 or more bases in length, up to alength that is one base shorter than the full length sequence.

As used herein the terms “pathological expression” and “pathogenicexpression”, and similar phrases, will together be referred to as“pathological expression”, and relate to differential expression of agene which is associated with a pathogenic state or a pathologicalcondition. Pathological expression thus relates to expression of a genethat differs from the expression level found in a non-diseasedcondition, or a non-pathological condition. In the present disclosure,pathological expression relates especially to gene identified as atarget gene, i.e., a gene that is a target for RNAi therapy. Thus,although pathological expression may generally relate to bothoverexpression of a gene and underexpression of a gene, the pathologicalexpression of a gene to be targeted by RNAi therapy is generallyoverexpression, and the RNAi therapy is intended to inhibit or reducethe overexpression.

A full-length gene or RNA further encompasses any naturally occurringsplice variants, allelic variants, other alternative transcripts, splicevariants that exhibit the same or a similar function as the naturallyoccurring full length gene, and the resulting RNA molecules. A fragmentof a gene can be any portion from the gene, which may or may notrepresent a functional domain, for example, a catalytic domain, a DNAbinding domain, etc.

“Complementary DNA” (cDNA), is a single-stranded DNA molecule that iscopied from an mRNA template by the enzyme reverse transcriptase,resulting in a sequence complementary to that of the mRNA. Those skilledin the an also use the term “cDNA” to refer to a double-stranded DNAmolecule that comprises such a single-stranded DNA molecule and itscomplementary DNA strand.

The term “operably linked” and similar terms and phrases are used todescribe the connection between regulatory elements and a gene or itscoding region. That is, gene expression is typically governed by certaintranscriptional regulatory elements, including constitutive or induciblepromoters, tissue-specific regulatory elements, and enhancers. Such agene or coding region is then said to be “operably linked to” or“operatively linked to” or “operably associated with” the regulatoryelements, meaning that the gene or coding region is controlled orinfluenced by the regulatory element.

As used herein the terms “interfere”, “silence” and “inhibit theexpression of”, and similar terms and phrases, in as far as they referto a target gene, relate to suppression or inhibition of expression of atarget either partially or essentially completely. Frequently suchinterference is manifested as a suppressed phenotype. In various casesexpression of the target gene is suppressed by at least about 10%, orabout 20%, or about 30%, or about 40%, or about 50%, or about 60%, orabout 70%, or about 80% by, administration of a targeting polynucleotideof the invention. In favorable embodiments, the target gene issuppressed by at least about 85%, or about 90%, or about 95%, orsubstantially completely, by administering a targeting polynucleotide.Such interference may be manifested in cells in a cell culture, or in atissue explant, or in vivo in a subject.

As used herein, the term “treatment” and similar terms and phrasesrelate to the application or administration of a therapeutic agent to asubject having a disease or condition, a symptom of disease, or apredisposition toward a disease, or application or administration of atherapeutic agent to an isolated tissue or cell line from the subject.Treatment is intended to promote curing or healing thereof, or toalleviate, relieve, alter, remedy, ameliorate, improve, or affect thedisease, the symptoms of disease, or the predisposition toward disease.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment of a disease, or an effectproviding prevention or diminishing the severity of the disease,respectively. The specific amount that is therapeutically effective canbe readily determined by an ordinary medical practitioner employingassessment of response in a treated subject, and may vary depending onfactors known in the art, such as the nature of the disease, thesubject's history and age, the stage of disease, and the administrationof other therapeutic agents.

As used herein, a “pharmaceutical composition” relates to a compositionthat includes a pharmacologically effective amount of a targetingpolynucleotide and a pharmaceutically acceptable carrier. As usedherein, “pharmacologically effective amount,” “therapeutically effectiveamount” or simply “effective amount” refers to that amount of aninhibitory polynucleotide effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta minimal measurable change in a clinical parameter associated with adisease or disorder, a therapeutically effective amount of a drug forthe treatment of that disease or disorder is the amount necessary toeffect at least extent of change in the parameter.

The term “pharmaceutically acceptable carrier” refers to a compositionfor administration of a therapeutic agent that is at least bothphysiologically acceptable and approvable by a regulatory agency.

Nucleotides may also be modified to harbor a label. Nucleotides bearinga fluorescent label or a biotin label, for example, are available fromSigma (St. Louis, Mo.).

RNA Interference

According to the invention, gene expression of targets in cancer cellsthat promote proliferation and/or metastasis is attenuated by RNAinterference. In particular, genes targeted in the present inventioninclude those designated ICT-1052, ICT-1053, ICT-1027, ICT-1051,ICT-1054, ICT-1020, ICT-1021 and ICT-1022. Transcription products of aTarget Gene are targeted within a cell by specific double stranded siRNAnucleotide sequences that are complementary to at least a segment of thetarget that contains any number of nucleotides between 15 and 30, or inmany cases, contains anywhere between 21 and 25 nucleotides. The targetmay occur in the 5′ untranslated (UT) region, in a coding sequence, orin the 3′ UT region. See, e.g., PCT applications WO00/44895, WO99/32619,WO01/75164, WO01/92513, WO01/29058, WO01/89304, WO02/16620, andWO02/29858, each incorporated by reference herein in their entirety.

According to the methods of the present invention, cancer cell geneexpression, and thereby cancer cell replication, is suppressed usingsiRNA. A targeting polynucleotide according to the invention includes ansiRNA oligonucleotide. An siRNA can be prepared by chemical synthesis ofnucleotide sequences identical or similar to a cancer cell targetsequence. See, e.g., Tuschl, Zamore, Lehmann, Bartel and Sharp (1999),Genes & Dev. 13: 3191-3197, incorporated herein by reference in itsentirety. Alternatively, a targeting siRNA can be obtained using atargeting polynucleotide sequence, for example, by digesting a cancercell ribopolynucleotide sequence in a cell-free system, such as but notlimited to a Drosophila extract, or by transcription of recombinantdouble stranded cancer cell cRNA.

Efficient silencing is generally observed with siRNA duplexes composedof a 15-30 nt strand complementary (i.e. antisense) to the chosen targetsequence and a 15-30 nt sense strand of the same length. In manyembodiments each strand of an siRNA paired duplex has in addition anoverhang of 1, 2, 3, or 4 unpaired nucleotides at the 3′ end. In commonembodiments the size of the overhang is 2 nt. The sequence of the 3′overhang makes an additional small contribution to the specificity ofsiRNA target recognition. In one embodiment, the nucleotides in the 3′overhang are ribonucleotides. In an alternative embodiment, thenucleotides in the 3′ overhang are deoxyribonucleotides. Use of 3′deoxynucleotides in a 3′ overhang provides enhanced intracellularstability.

A recombinant expression vector of the invention that includes atargeting sequence, when introduced within a cell, is processed toprovide an RNA that includes an siRNA sequence targeting a gene in acancer cell implicated in cell proliferation and/or metastasis. Such avector is a DNA molecule cloned into an expression vector comprisingoperatively-linked regulatory sequences flanking the cancer celltargeting sequence in a manner that allows for expression of thetargeting sequence. From the vector, an RNA molecule that is antisenseto cancer cell RNA is transcribed by a first promoter (e.g., a promotersequence 3′ of the cloned DNA) and an RNA molecule that is the sensestrand for the cancer cell RNA target is transcribed by a secondpromoter (e.g., a promoter sequence 5′ of the cloned DNA). The sense andantisense strands then hybridize in vivo to generate siRNA constructstargeting the cancer cell RNA molecule for silencing of the gene.Alternatively, two separate constructs can be utilized to create thesense and anti-sense strands of a siRNA construct. Further, cloned DNAcan encode a transcript having a hairpin secondary structure, wherein asingle transcript has both the sense and complementary antisensesequences from the target gene or genes. In an example of thisembodiment, a hairpin RNAi transcription product includes a firstsequence that is similar to all or a portion of the target gene and asecond sequence complementary to the first sequence, so disposed as toform a hairpin duplex. In another example, a hairpin RNAi product is asiRNA. The regulatory sequences flanking the cancer cell sequence in thevector may be identical or may be different, such that their expressionmay be modulated independently, or in a temporal or spatial manner.

In certain embodiments, siRNAs are transcribed intracellularly bycloning the cancer cell Target Gene templates into a vector containing,e.g., a RNA poi III transcription unit from the smaller nuclear RNA(snRNA) U6 or the human RNase P RNA H1. One example of a vector systemis the GeneSuppressor™ RNA Interference kit (commercially available fromImgenex). The U6 and H1 promoters are members of the type III class ofPol III promoters. The +1 nucleotide of the U6-like promoters is alwaysguanosine, whereas the +1 for H1 promoters is adenosine. The terminationsignal for these promoters is defined by five consecutive thymidines.The transcript is typically cleaved after the second uridine. Cleavageat this position generates a 3′ UU overhang in the expressed siRNA,which is similar to the 3′ overhangs of synthetic siRNAs. Any sequenceless than 400 nucleotides in length can be transcribed by thesepromoters, therefore they are ideally suited for the expression ofaround 21-nucleotide siRNAs in, e.g., an approximately 50-nucleotide RNAhairpin-loop transcript. An initial BLAST homology search for theselected siRNA sequence is done against an available nucleotide sequencelibrary to ensure that only an intended target preferentially expressedin a cancer cell, but no nontargeted host gene, is identified. See,Elbashir et al. 2001 EMBO J. 20(23):6877-88.

Synthesis of Polynucleotides

Oligonucleotides corresponding to targeting nucleotide sequences, andpolynucleotides that include targeting sequences, can be prepared bystandard synthetic techniques, e.g., using an automated DNA synthesizer.Methods for synthesizing oligonucleotides include well-known chemicalprocesses, including, but not limited to, sequential addition ofnucleotide phosphoramidites onto surface-derivatized particles, asdescribed by T. Brown and Dorcas J. S. Brown in Oligonucleotides andAnalogues A Practical Approach, F. Eckstein, editor, Oxford UniversityPress, Oxford, pp. 1-24 (1991), and incorporated herein by reference.

An example of a synthetic procedure uses Expedite RNA phosphoramiditesand thymidine phosphoramidite (Proligo, Germany). Syntheticoligonucleotides are deprotected and gel-purified (Elbashir et al.(2001) Genes & Dev. 15, 188-200), followed by Sep-Pak C18 cartridge(Waters, Milford, Mass., USA) purification (Tuschl et al. (1993)Biochemistry, 32:11658-11668). Complementary ssRNAs are incubated in anannealing buffer (100 mM potassium acetate, 30 mM HEPES-KOH at pH 7.4, 2mM magnesium acetate) for 1 min at 90° C. followed by 1 h at 37° C. tohybridize to the corresponding ds-siRNAs.

Other methods of oligonucleotide synthesis include, but are not limitedto solid-phase oligonucleotide synthesis according to thephosphotriester and phosphodiester methods (Narang, et al., (1979) Meth.Enzymol. 68:90), and to the H-phosphonate method (Garegg, P. J., et al.,(1985) “Formation of internucleotidic bonds via phosphonateintermediates”, Chem. Scripta 25, 280-282; and Froehler, B. C., et al.,(1986a) “Synthesis of DNA via deoxynucleoside H-phosphonateintermediates”, Nucleic Acid Res., 14, 5399-5407, among others) andsynthesis on a support (Beaucage, et al. (1981) Tetrahedron Letters22:1859-1862) as well as phosphoramidate techniques (Caruthers, M. H.,et al., “Methods in Enzymology,” Vol. 154, pp. 287-314 (1988), U.S. Pat.Nos. 5,153,319, 5,132,418, 4,500,707, 4,458,066, 4,973,679, 4,668,777,and 4,415,732, and others described in “Synthesis and Applications ofDNA and RNA,” S. A. Narang, editor, Academic Press, New York, 1987, andthe references contained therein, and nonphosphoramidite techniques.Solid phase synthesis helps isolate the oligonucleotide from impuritiesand excess reagents. Once cleaved from the solid support theoligonucleotide may be further isolated by known techniques.

Inhibitory Polynucleotides of the Invention

A targeting polynucleotide of the invention may be a DNA, an RNA, amixed DNA-RNA polynucleotide strand, or a DNA-RNA hybrid. An example ofthe latter is an RNA sequence terminated at the 3′ end with adeoxydinucleotide sequence, such as d(TT), d(UU), d(TU), d(UT), as wellas other possible dinucleotides. In additional embodiments the 3′overhang may be constituted of ribonucleotides having the basesspecified above, or others. Furthermore, the oligonucleotidepharmaceutical agent may be single stranded or double stranded. Severalembodiments of the therapeutic oligonucleotides of the invention areenvisioned to be oligoribonucleotides, or oligoribonucleotides with 3′d(TT) terminals. A single stranded targeting polynucleotide, ifadministered into a mammalian cell, is readily converted upon entry to adouble stranded molecule by endogenous enzyme activity resident in thecell. The resulting double stranded oligonucleotide triggers RNAinterference.

The targeting polynucleotide may be a single stranded polynucleotide ora double stranded polynucleotide. A targeting nucleotide sequencecontained within the polynucleotide may be comprised entirely ofnaturally occurring nucleotides, or at least one nucleotide of thepolynucleotide may be a modified nucleotide or a derivatized nucleotide.Modification or derivatization may accomplish objectives such asstabilization of the polynucleotide, optimizing the hybridization of astrand with a complement, or enhancing the induction of the RNAiprocess. All equivalent polynucleotides that are understood by workersof skill in molecular biology, cell biology, oncology and related fieldsof medicine, and other fields related to the present invention, tocomprise a targeting sequence are within the scope of the presentinvention.

A polynucleotide of the invention includes a targeting sequence, and iseffective to inhibit the growth or replication of cells characteristicof the disease or pathology. The first nucleotide sequence, or targetingsequence, in important embodiments of the invention, may be at least 15nucleotides (nt) in length, and at most 100 nt. In certain importantembodiments, the length may be at most 70 nt. In still more importantembodiments, the first nucleotide sequence may be 15 nt, or 16 nt, or 17nt, or 18 nt, or 19 nt, or 20 nt, or 21 nt, or 22 nt, or 23 nt, or 24nt, or 25 nt, or 26 nt, or 27 nt, or 28 nt, or 29 nt, or 30 nt inlength.

The first targeting nucleotide sequence or its complement is generallyat least 80% complementary to the sequence that it is targeting in thetarget gene. Thus in those embodiments identified in the precedingparagraph in which the target sequence ranges between 15 and 30 nt inlength, no more than 3, or 4, or 5 nucleotides may differ fromcomplementarity with the target sequence. In significant embodiments thefirst nucleotide sequence or its complement is at least 85%complementary, or at least 90% complementary, or at least 95%complementary, or at least 97% complementary, to the target sequence.

The first nucleotide sequence or its complement is sufficientlycomplementary to its target sequence that it induces the RNAinterference phenomenon, thereby promoting cleavage of the targetnucleic acid by RNase activity. Any equivalent first nucleotide sequencepromoting cleavage of the pathogenic nucleic acid falls within the scopeof the present invention.

A short hairpin RNA (shRNA) is contemplated as being comprised in thefirst polynucleotide of the invention. A shRNA includes a targetingfirst nucleotide sequence, an intervening loop-forming nucleotidesequence, and a second targeting nucleotide sequence complementary tothe first targeting sequence. Without wishing to be bound by theory, itis believed that a polynucleotide comprising a first target sequence, aloop, and a second target sequence complementary to the first loopsaround to form an intramolecular double stranded “hairpin” structure inwhich the second complementary sequence hybridizes with the first targetsequence. Again, not wishing to be bound by theory, it is believed thatthe RNAi phenomenon is induced by a double stranded RNA sequence forminga complex with its target sequence. Use of a shRNA affords an optimalmeans to provide the double stranded targeting polynucleotide effectiveto silence the targeted gene.

In important embodiments the targeting polynucleotide additionallyincludes a promoter and/or an enhancer sequence in operable relationshipwith the first nucleotide sequence, or, in the case of an shRNA, inoperable relationship with the entire shRNA construct including thefirst nucleotide sequence, the loop, and the complementary nucleotidesequence.

Vectors. The present invention provides various vectors that contain oneor more first polynucleotides of the invention. By including more thanone first polynucleotide the vector carries targeting sequences directedat more than one pathogenic target sequence. The pathogenic targetsequences may be directed to the same gene, or to different genes in thecells of a subject suffering from the pathology. Advantageously anyvector of the invention includes a promoter, an enhancer, or both,operably linked to the first nucleotide sequence or to the shRNAsequence, respectively.

Methods for preparing the vectors of the invention are widely known inthe fields of molecular biology, cell biology, oncology and relatedfields of medicine, and other fields related to the present invention.Methods useful for preparing the vectors are described, by way onnonlimiting example, in Molecular Cloning: A Laboratory Manual (3^(rd)Edition) (Sambrook, J et al. (2001) Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y.), and Short protocols in molecular biology(5^(th) Ed.) (Ausubel F M et al. (2002) John Wiley & Sons, New YorkCity).

Antibodies

The term “antibody” as used herein refers to immunoglobulin moleculesand immunologically active portions of immunoglobulin (Ig) molecules,i.e., molecules that contain an antigen binding site that specificallybinds (immunoreacts with) an antigen. Such antibodies include, but arenot limited to, polyclonal, monoclonal, chimeric, single chain, F_(ab),F_(ab′), and F_((ab′)2) fragments, and an F_(ab) expression library. Ingeneral, antibody molecules obtained from humans relates to any of theclasses IgG, IgM, IgA, IgE and IgD, which differ from one another by thenature of the heavy chain present in the molecule. Certain classes havesubclasses as well, such as IgG₁, IgG₂, and others. Furthermore, inhumans, the light chain may be a kappa chain or a lambda chain.Reference herein to antibodies includes a reference to all such classes,subclasses and types of human antibody species.

An isolated protein of the invention intended to serve as an antigen, ora portion or fragment thereof can be used as an immunogen to generateantibodies that immunospecifically bind the antigen, using standardtechniques for polyclonal and monoclonal antibody preparation. Thefull-length protein can be used or, alternatively, the inventionprovides antigenic peptide fragments of the antigen for use asimmunogens. An antigenic peptide fragment comprises at least 6 aminoacid residues of the amino acid sequence of the full length protein, andencompasses an epitope thereof such that an antibody raised against thepeptide forms a specific immune complex with the full length protein orwith any fragment that contains the epitope. Preferably, the antigenicpeptide comprises at least 10 amino acid residues, or at least 15 aminoacid residues, or at least 20 amino acid residues, or at least 30 aminoacid residues. Preferred epitopes encompassed by the antigenic peptideare regions of the protein that are located on its surface; commonlythese are hydrophilic regions.

In certain embodiments of the invention, at least one epitope of aTarget Gene polypeptide encompassed by the antigenic peptide is a regionof a polypeptide that is located on the surface of the protein, e.g., ahydrophilic region. A hydrophobicity analysis of the human proteinsequence will indicate which regions of a polypeptide are particularlyhydrophilic and, therefore, are likely to encode surface residues usefulfor targeting antibody production. As a means for targeting antibodyproduction, hydropathy plots showing regions of hydrophilicity andhydrophobicity may be generated by any method well known in the art,including, for example, the Kyte Doolittle or the Hopp Woods methods,either with or without Fourier transformation. See, e.g., Hopp andWoods, 1981, Proc. Nat. Acad. Sci. USA 78: 3824-3828; Kyte and Doolittle1982, J. Mol. Biol. 157: 105-142, each incorporated herein by referencein their entirety. Antibodies that are specific for one or more domainswithin an antigenic protein, or derivatives, fragments, analogs orhomologs thereof, are also provided herein.

Various procedures known within the art may be used for the productionof polyclonal or monoclonal antibodies directed against a protein of theinvention, or against derivatives, fragments, analogs homologs ororthologs thereof (see, for example, Antibodies: A Laboratory Manual,Harlow E, and Lane D, 1988, Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y., incorporated herein by reference). Some of theseantibodies are discussed below.

Polyclonal Antibodies

For the production of polyclonal antibodies, various suitable hostanimals (e.g., rabbit, goat, mouse or other mammal) may be immunized byone or more injections with the native protein, a synthetic variantthereof, or a derivative of the foregoing. An appropriate immunogenicpreparation can contain, for example, the naturally occurringimmunogenic protein, a chemically synthesized polypeptide representingthe immunogenic protein, or a recombinantly expressed immunogenicprotein. Furthermore, the protein may be conjugated to a second proteinknown to be immunogenic in the mammal being immunized. Examples of suchimmunogenic proteins include but are not limited to keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsininhibitor. The preparation can further include an adjuvant. Variousadjuvants used to increase the immunological response include, but arenot limited to, Freund's (complete and incomplete), mineral gels (e.g.,aluminum hydroxide), surface active substances (e.g., lysolecithin,pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol,etc.), adjuvants usable in humans such as Bacille Calmette-Guerin andCorynebacterium parvum, or similar immunostimulatory agents. Additionalexamples of adjuvants which can be employed include MPL-TDM adjuvant(monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).

Monoclonal Antibodies

The term “monoclonal antibody” (MAb) or “monoclonal antibodycomposition”, as used herein, refers to a population of antibodymolecules that contain only one molecular species of antibody moleculeconsisting of a unique light chain gene product and a unique heavy chaingene product. In particular, the complementarity determining regions(CDRs) of the monoclonal antibody are identical in all the molecules ofthe population. MAbs thus contain an antigen binding site capable ofimmunoreacting with a particular epitope of the antigen characterized bya unique binding affinity for it.

Monoclonal antibodies can be prepared using hybridoma methods, such asthose described by Kohler and Milstein, Nature, 256:495 (1975). In ahybridoma method, a mouse, hamster, or other appropriate host animal, istypically immunized with an immunizing agent to elicit lymphocytes thatproduce or are capable of producing antibodies that will specificallybind to the immunizing agent. Alternatively, the lymphocytes can beimmunized in vitro.

The immunizing agent will typically include the protein antigen, afragment thereof or a fusion protein thereof. Generally, eitherperipheral blood lymphocytes are used if cells of human origin aredesired, or spleen cells or lymph node cells are used if non-humanmammalian sources are desired. The lymphocytes are then fused with animmortalized cell line using a suitable fusing agent, such aspolyethylene glycol, to form a hybridoma cell [Goding, MonoclonalAntibodies: Principles and Practice. Academic Press, (1986) pp. 59-103].Immortalized cell lines are usually transformed mammalian cells,particularly myeloma cells of rodent, bovine and human origin. Usually,rat or mouse myeloma cell lines are employed. The hybridoma cells can becultured in a suitable culture medium that preferably contains one ormore substances that inhibit the growth or survival of the unfused,immortalized cells. For example, if the parental cells lack the enzymehypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), theculture medium for the hybridomas typically will include hypoxanthine,aminopterin, and thymidine (“HAT medium”), which substances prevent thegrowth of HGPRT-deficient cells.

The monoclonal antibodies can also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567. DNA encoding themonoclonal antibodies of the invention can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells of theinvention serve as a preferred source of such DNA. Once isolated, theDNA can be placed into expression vectors, which are then transfectedinto host cells such as simian COS cells, Chinese hamster ovary (CHO)cells, or myeloma cells that do not otherwise produce immunoglobulinprotein, to obtain the synthesis of monoclonal antibodies in therecombinant host cells.

Humanized Antibodies

The antibodies directed against the protein antigens of the inventioncan further comprise humanized antibodies or human antibodies. Theseantibodies are suitable for administration to humans without engenderingan immune response by the human against the administered immunoglobulin.Humanized forms of antibodies are chimeric immunoglobulins,immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab′,F(ab′)₂ or other antigen-binding subsequences of antibodies) that areprincipally comprised of the sequence of a human immunoglobulin, andcontain minimal sequence derived from a non-human immunoglobulin.Humanization can be performed following the method of Winter andco-workers (Jones et al., Nature, 321:522-525 (1986); Riechmann et al.,Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536(1988)), by substituting rodent CDRs or CDR sequences for thecorresponding sequences of a human antibody. (See also U.S. Pat. No.5,225,539.) The humanized antibody optimally also will comprise at leasta portion of an immunoglobulin constant region (Fc), typically that of ahuman immunoglobulin (Jones et al., 1986; Riechmann et al., 1988; andPresta, Curr. Op. Struct. Biol., 2:593-596 (1992)).

Human Antibodies

Fully human antibodies essentially relate to antibody molecules in whichthe entire sequence of both the light chain and the heavy chain,including the CDRs, arise from human genes. Such antibodies are termed“human antibodies”, or “fully human antibodies” herein. Human monoclonalantibodies can be prepared by the trioma technique; the human B-cellhybridoma technique (see Kozbor, et al., 1983 Immunol Today 4: 72) andthe EBV hybridoma technique to produce human monoclonal antibodies (seeCole, et al., 1985 In: MONOCLONAL ANTIBODIES AND CANCER THERAPY, Alan R.Liss, Inc., pp. 77-96). Human monoclonal antibodies may be utilized inthe practice of the present invention and may be produced by using humanhybridomas (see Cote, et al., 1983. Proc Natl Acad Sci USA 80:2026-2030) or by transforming human B-cells with Epstein Barr Virus invitro (see Cole, et al. In: MONOCLONAL ANTIBODIES AND CANCER THERAPY,Alan R. Liss, Inc., pp. 77-96).

In addition, human antibodies can also be produced using additionaltechniques, including phage display libraries (Hoogenboom and Winter, J.Mol. Biol. 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)).Similarly, human antibodies can be made by introducing humanimmunoglobulin loci into transgenic animals, e.g., mice in which theendogenous immunoglobulin genes have been partially or completelyinactivated. Upon challenge, human antibody production is observed,which closely resembles that seen in humans in all respects, includinggene rearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in Marks et al.(Bio/Technology 10, 779-783 (1992)); Lonberg et al. (Nature 368 856-859(1994)); Morrison (Nature 368, 812-13 (1994)); Fishwild et al, (NatureBiotechnology 14, 845-51 (1996)); Neuberger (Nature Biotechnology 14,826 (1996)); and Lonberg and Huszar (Intern. Rev. Immunol. 13 65-93(1995)).

Human antibodies may additionally be produced using transgenic nonhumananimals which are modified so as to produce fully human antibodiesrather than the animal's endogenous antibodies in response to challengeby an antigen. (See PCT publication WO94/02602). The endogenous genesencoding the heavy and light immunoglobulin chains in the nonhuman hosthave been incapacitated, and active loci encoding human heavy and lightchain immunoglobulins are inserted into the host's genome. The humangenes are incorporated, for example, using yeast artificial chromosomescontaining the requisite human DNA segments. An animal which providesall the desired modifications is then obtained as progeny bycrossbreeding intermediate transgenic animals containing fewer than thefull complement of the modifications. The preferred embodiment of such anonhuman animal is a mouse, and is termed the Xenomouse™ as disclosed inPCT publications WO 96/33735 and WO 96/34096.

F_(ab) Fragments and Single Chain Antibodies

According to the invention, techniques can be adapted for the productionof single-chain antibodies specific to an antigenic protein of theinvention (see e.g., U.S. Pat. No. 4,946,778). In addition, methods canbe adapted for the construction of F_(ab) expression libraries (seee.g., Huse, et al., 1989 Science 246: 1275-1281) to allow rapid andeffective identification of monoclonal F_(ab) fragments with the desiredspecificity for a protein or derivatives, fragments, analogs or homologsthereof. Antibody fragments that contain the idiotypes to a proteinantigen may be produced by techniques known in the art including, butnot limited to: (i) an F_((ab′)2) fragment produced by pepsin digestionof an antibody molecule; (ii) an F_(ab) fragment generated by reducingthe disulfide bridges of an F_((ab′)2) fragment; (iii) an F_(ab)fragment generated by the treatment of the antibody molecule with papainand a reducing agent and (iv) F_(v) fragments.

Antibody Therapeutics

Antibodies of the invention, including polyclonal, monoclonal, humanizedand fully human antibodies, may used as therapeutic agents. Such agentswill generally be employed to treat or prevent a disease or pathology ina subject. An antibody preparation, preferably one having highspecificity and high affinity for its target antigen, is administered tothe subject and will generally have an effect due to its binding withthe target. Such an effect may be one of two kinds, depending on thespecific nature of the interaction between the given antibody moleculeand the target antigen in question. In the first instance,administration of the antibody may abrogate or inhibit the binding ofthe target with an endogenous ligand to which it naturally binds. Inthis case, the antibody binds to the target and masks a binding site ofthe naturally occurring ligand, wherein the ligand serves as an effectormolecule. Thus the receptor mediates a signal transduction pathway forwhich ligand is responsible.

Alternatively, the effect may be one in which the antibody elicits aphysiological result by virtue of binding to an effector binding site onthe target molecule. In this case the target, a receptor having anendogenous ligand which may be absent or defective in the disease orpathology, binds the antibody as a surrogate effector ligand, initiatinga receptor-base.

Pharmaceutical Compositions of Antibodies

Antibodies specifically binding a protein of the invention, as well asother molecules identified by the screening assays disclosed herein, canbe administered for the treatment of various disorders in the form ofpharmaceutical compositions. Principles and considerations involved inpreparing such compositions, as well as guidance in the choice ofcomponents are provided, for example, in Remington: The Science AndPractice Of Pharmacy 19th ed. (Alfonso R. Gennaro, et, al., editors)Mack Pub. Co., Easton, Pa.: 1995; Drug Absorption Enhancement: Concepts,Possibilities, imitations, And Trends, Harwood Academic Publishers,Langhorne, Pa., 1994; and Peptide And Protein Drug Delivery (Advances InParenteral Sciences, Vol. 4), 1991, M. Dekker, New York.

The present invention includes antibodies binding to Target Gene proteinproducts that can be produced from mouse, rabbit, goat, horse and othermammals. Therapeutic antibodies directed product polypeptides of anICT-1053 gene, or an ICT-1052 gene, or an ICT-1027 gene, or an ICT-1051gene, or an ICT-1054 gene, or an ICT-1020 gene, or an ICT-1021 gene, oran ICT-1022 gene include the mouse antibodies, chimeric antibodies,humanized forms and human monoclonal antibodies. Specific monoclonalantibodies directed against a Target Gene product polypeptide are ableto increase the apoptosis activity of cancer cells when they were treatwith the antibody. Such specific monoclonal antibodies are further ableto decrease cancer cell proliferation when they are treated with theantibody. Target Gene specific monoclonal antibodies have potential toinhibit tumor growth in vivo, with both xenograft and Syngenic tumormodels.

Cancer Cell Target Polynucleotides of the Invention

Several target genes were identified as lead target genes in the presentinvention. These target genes are considered validated targets forinhibition of tumor growth, disease progression and methods andcompositions for the inhibition and treatment of tumors and cancers inmammals, and in particular, in humans. The validation is based in parton the showings presented in the Examples below of that the Target.Genes are targets for inhibition of tumor growth or promotion ofapoptosis, and can thus be used as targets for therapy; and, they alsocan be used to identify compounds useful in the diagnosis, prevention,and therapy of tumors and cancers. The Target Genes are summarized inTable 1.

TABLE 1 Target Genes SEQ ID SEQ ID NO: for NO: for Target Polynu-Encoded Gene GenBank Acc. Nos. Characterization cleotide PolypeptideICT-1052 J02958, NM_000245, H. sap. c-Met, met proto- 1 2 NM_008591,AF075090, oncogene (hepatocyte and X54559 growth factor (HGF) receptor)ICT-1053 BC002506, NM_007217, H. sap. programmed cell 3 4 BC019650,NM_019745, death 10 (PDCD10) BC016353, NM_145860, NM_145859, andAF022385 ICT-1027 BC043007, AF171699, H. sap. growth factor 5 6NM_002086, receptor-bound protein 2, NM_203506, CR450363, GRB2 CR541942,and M96995 ICT-1051 L24038, U01337, H. sap. murine sarcoma Z84466,AB208831, 3611 viral (v-raf) oncogene AK130043, BC002466, homolog 1(ARAF1)), BC007514, BT019864, (Ser/Thr protein kinase) M13829, X04790ICT-1054 BC053586, BC050597, H. sap. programmed cell BC045555, BC044220,death 6 BC028242, BC020552, BC019918, BC014604, BC012384, AK223366,AF035606, AK001917 ICT-1020 AJ132261, BC046934, H. sap. hypotheticalNM_177438, AB028449 helicase K12H4.8-like protein; H. sap. Ortholog ofDrosophila Dicer ICT-1021 BC020690, AB018549, H. sap. lymphocyte antigenAF168121 96, MD-2, ESOP1 ICT-1022 BC069309, BC069397, H. sap. G antigen2 BC069558, U19143 (GAGE-2)

ICT-1052: The target ICT-1052 has been identified as C-METproto-oncogene (hepatocyte growth factor (HGF) receptor; see Bottaro etal., Science, 251:802-804; Naldini et al, Oncogene, 6: 501-504; Park etal., 1987, Proc. Natl. Acad. USA, 84: 6379-83; WO 92/13097; WO 93/15754;WO 92/20792; Prat et al., 1991, Mol. Cell. Biol., 11:5954-62). Theexpression of c-Met is detected in various human solid tumors (Prat etal., 1991, Int. J. cancer, 49:323-328) and is implicated in thyroidtumors derived from follicular epithelium (DiRenzo et al., 1992,Oncogene, 7:2549-53). c-Met and its splice variants behaved like a tumorenhancing target since the siRNA-mediated ICT-1052 knockdown resulted intumor growth inhibition. It is believed that the target ICT-1052 is atumor stimulator and so is a target for treating tumors, cancers, andprecancerous states in mammalian tissues using antibodies, smallmolecules, antisense, siRNA and other antagonist agents.

Several antibodies to c-Met, including monoclonal antibodies (mAbs),referred to as DL-21, DN-30, DN-31, and DO-24, are specific for theextracellular domain of the 145-kDa β-chain of the c-Met (WO 92/20792;Prat et al., 1991, Mol. Cell. Biol., 11:5954-62) or the intracellulardomain (Bottaro et al, Science, 251:802-804). Such antibodies have beenused in diagnostic and therapeutic applications (Prat et al., 1991, Int.J. Cancer, 49:323-328; Yamada, et al., 1994, Brain Research,637:308-312; Crepaldi et al., 1994, J. Cell Biol., 125; 313-20; U.S.Pat. No. 5,686,292; U.S. Pat. No. 6,207,152; patent application of MarkKay; US Patent Application Publication 20030118587; WO2004/07877 and WO2004/072117).

The target ICT-1052 includes polymorphic variants, alleles, mutants, andinterspecies orthologs that have (i) substantial nucleotide sequencehomology (for example, at least 60% identity, preferably at least 70%sequence identity, more preferably at least 80%, still more preferablyat least 90% and even more preferably at least 95%) with the nucleotidesequence of the sequence disclosed in the GenBank accession nos.referenced in Table 1, or to its encoded polypeptide. ICT-1052polynucleotides or polypeptides are typically from a mammal including,but not limited to, human, rat, mouse, hamster, cow, pig, horse, andsheep.

A nucleotide sequence for ICT-1052 contains 6641 base pairs (see SEQ IDNO:1 in the Sequence Listing appended hereto; disclosed in prioritydocument U.S. 60/642,067), encoding a protein of 1390 amino acids (seeSEQ ID NO:2 in the Sequence Listing appended hereto; disclosed inpriority document U.S. 60/642,067).

ICT-1053: The target designated ICT-1053 is PDCD10, programmed celldeath 10 (PDCD10. This gene encodes a protein, originally identified ina premyeloid cell line, with similarity to proteins that participate inapoptosis. PDCD10 protein was able to inhibit the apoptosis of 293 cellsin culture (Ma et al. 1998). ICT-1053 is up regulated in fast growingtumors. Inhibition of this target can play an important role in thetherapy of various cancer types, tumors and precancerous states. ThussiRNA, monoclonal antibody, and small molecule inhibitors of this targetmay be useful for cancer treatment using antibodies, small molecules,antisense, siRNA and other antagonist agents.

The target ICT-1053 includes polymorphic variants, alleles, mutants, andinterspecies orthologs that have (i) substantial nucleotide sequencehomology (for example, at least 60% identity, preferably at least 70%sequence identity, more preferably at least 80%, still more preferablyat least 90% and even more preferably at least 95%) with the nucleotidesequence of the sequence disclosed in the GenBank accession nos.referenced in Table 1, or to its encoded polypeptide. ICT-1053polynucleotides or polypeptides are typically from a mammal including,but not limited to, human, rat, mouse, hamster, cow, pig, horse, andsheep.

A nucleotide sequence for ICT-1053 contains 1466 base pairs (see SEQ IDNO:3 in the Sequence Listing appended hereto; disclosed in prioritydocument U.S. 60/642,067), encoding a protein of 212 amino acids (seeSEQ ID NO:4 in the Sequence Listing appended hereto; disclosed inpriority document U.S. 60/642,067).

ICT-1027: The target ICT-1027 is Homo sapiens growth factorreceptor-bound protein 2, GRB2, having the ability to bind the epidermalgrowth factor receptor (EGFR) (Lowenstein et al. (1992)). GRB2 geneencodes a protein that has homology to noncatalytic regions of the SRConcogene product, and is a homolog of the Sem5 gene of C. elegans, whichis involved in the signal transduction pathway leading to induction ofvulva formation. Drk, the Drosophila homolog of GRB2, plays an essentialrole in fly photoreceptor development. Various studies have providedevidence for a mammalian GRB2-Ras signaling pathway, mediated by SH2/SH3domain interactions, that has multiple functions in embryogenesis andcancer. ICT-1027 is up regulated in fast growing tumors. It is believedthat the target ICT-1027 is a novel target for treating tumors, cancers,and precancerous states in mammalian tissues using antibodies, smallmolecules, antisense, siRNA and other antagonist agents.

The target ICT-1027 includes polymorphic variants, alleles, mutants, andinterspecies orthologs that have (i) substantial nucleotide sequencehomology (for example, at least 60% identity, preferably at least 70%sequence identity, more preferably at least 80%, still more preferablyat least 90% and even more preferably at least 95%) with the nucleotidesequence of the sequence disclosed in the GenBank accession nos.referenced in Table 1, or to its encoded polypeptide. ICT-1052polynucleotides or polypeptides are typically from a mammal including,but not limited to, human, rat, mouse, hamster, cow, pig, horse, andsheep.

A nucleotide sequence for ICT-1027 contains 3317 base pairs (see SEQ IDNO:5 in the Sequence Listing appended hereto; disclosed in prioritydocument U.S. 60/642,067), encoding a protein of 217 amino acids (seeSEQ ID NO:6 in the Sequence Listing appended hereto; disclosed inpriority document U.S. 60/642,067).

Target ICT-1051. Limiting Apaf-1 activity may alleviate bothpathological protein aggregation and neuronal cell death in HD. A-Rafresidues are identified that bind to specific phosphoinositides,possibly as a mechanism to localize the enzyme to particular membranemicrodomains rich in these phospholipids. The mutation analysis of theconserved regions in the ARAF gene in human colorectal adenocarcinomahas reviewed its role in tumorigenesis. In a two-hybrid screen of humanfetal liver cDNA library, TH1 was detected as a new interaction partnerof A-Raf; this specific interaction may have played a critical role inthe activation of A-Raf. A-Raf kinase is negatively regulated bytrihydrophobin 1 and A-Raf interacts with MEK1 and activates MEK1 byphosphorylation.

Target ICT-1054. Raf-1 may mediate its anti-apoptotic function byinterrupting ASK1-dependent phosphorylation of ALG-2 (PCDP6). Thedown-regulation of ALG-2 in human uveal melanoma cells compared to theirprogenitor cells, normal melanocytes, may provide melanoma cells with aselective advantage by interfering with Ca+-mediated apoptotic signals,thereby enhancing cell survival. Data show that ALG-2 is overexpressedin liver and lung neoplasms, and is mainly found in epithelial cells inthe lung. ALG-2 has roles in both cell proliferation and cell death. Thepenta-EF-hand domain of ALG-2 interacts with amino-terminal domains ofboth annexin VII and annexin XI in a Ca2+-dependent manner.Pro/Gly/Tyr/Ala-rich hydrophobic region in Anexin XI masked theCa(2+)-dependently exposed hydrophobic surface of ALG-2. ALG-2 isstabilized by dimerization through its fifth EF-hand region.Apoptosis-linked gene 2 binds to the death domain of Fas and dissociatesfrom Fas during Fas-mediated apoptosis in Jurkat cells.

Target ICT-1020. Various attributes of the 3′ end structure, includingoverhang length and sequence composition, play a primary role indetermining the position of Dicer cleavage in both dsRNA andunimolecular, short hairpin RNA. Dicer is essential for formation of theheterochromatin structure in vertebrate cells. Dicer has a single RNApost-transcriptional processing center. The fragile X syndrome CGGrepeats readily form RNA hairpins and is digested by the human Dicerenzyme, a step central to the RNA interference effect on geneexpression.

Target ICT-1021. There is evidence to illustrate the function of MD-2 asthe primary molecular site of lipopolysaccharide (LPS)-dependentantagonism of Escherichia coli LPS at the Toll-like receptor 4 signalingcomplex. These results clearly demonstrate that the amino-terminal TLR4region of Glu(24)-Pro(34) is critical for MD-2 binding and LPSsignaling. MD-2 is an important mediator of organ inflammation duringsepsis. A rare A to G substitution at position 103, encoding a mutationof Thr 35 to Ala, results in a reduced lipopolysaccharide-inducedsignaling. Results show that the N-terminal region of toll-like receptor4 is essential for association with MD-2, which is required for the cellsurface expression and hence the responsiveness to lipopolysaccharide.The extracellular toll-like receptor 4 (TLR4)domain-MD-2 complex iscapable of binding lipopolysaccharide (LPS) and attenuating LPS-inducedNF-kappa B activation and IL-8 secretion in wild-type TLR4-expressingcells. The regulation of MD-2 expression in airway epithelia andpulmonary macrophages may serve as a means to modify endotoxinresponsiveness in the airway. MD-2 basic amino acid clusters areinvolved in cellular lipopolysaccharide recognition TLR4 is able toundergo multiple glycosylations without MD-2 but that the specificglycosylation essential for cell surface expression requires thepresence of MD-2. Two functional domains exist in MD-2, one responsiblefor Toll-like receptor 4-binding and another that mediates theinteraction with the agonist (lipopolysaccharide). MD-2 binds tolipopolysaccharide, leading to Toll-like receptor-4 aggregation andsignal transduction. Some data support the hypothesis thatlipopolysaccharide binding protein can inhibit cell responses tolipopolysaccharide (LPS) by inhibiting LPS transfer from membrane CD14to the Toll-like receptor 4-MD-2 signaling receptor. Innate immunerecognition of LTA via LBP, CD14, and TLR-2 represents an importantmechanism in the pathogenesis of systemic complications in the course ofinfectious diseases brought about by Gram-positive pathogens while TLR-4and MD-2 are not involved. Disulfide bonds are involved in the assemblyand function of this protein. Lipopolysaccharide rapidly traffics to andfrom the Golgi apparatus with the toll-like receptor 4-MD-2-CD14complex. Expression regulated by immune-mediated signals in intestinalepithelial cells. MD-2 can confer on mouse Toll-like receptor 4 (TLR4)responsiveness to lipid A but not to lipid IVa, thus influencing thefine specificity of TLR4. Expression of accessory molecule MD-2 isdownregulated in intestinal epithelial cells by a mechanism which limitsdysregulated immune signaling and activation of proinflammatory genes inresponse to bacterial lipopolysaccharide. There is no previous report toshow that MD-2 is involved in the tumorigenesis.

Target ICT-1022. This gene belongs to a family of genes that areexpressed in a variety of tumors but not in normal tissues, except forthe testis. The sequences of the family members are highly related butdiffer by scattered nucleotide substitutions. The antigenic peptideYRPRPRRY, which is also encoded by several other family members, isrecognized by autologous cytolytic T lymphocytes. Nothing is presentlyknown about the function of this protein.

It is reported in the Examples below that when the targets ICT-1052,ICT-1053, ICT-1027, ICT-1051, ICT-1054, ICT-1020, ICT-1021 and ICT-1022were down regulated by two specific siRNA molecules tumor growth ratesdecreased.

The invention provides broadly for oligonucleotides intended to provokean RNA interference phenomenon upon entry into a precancerous orcancerous cell. The present invention, while not restricted in thenature of the cancer cell target gene, emphasizes oligonucleotidestargeting a Target Gene of the invention. RNA interference is engenderedwithin the cell by appropriate double stranded RNAs one of whose strandshas a complement that is identical to or highly similar to a sequence ina target polynucleotide of the cancer cell. In general, anoligonucleotide that targets a Target Gene may be a DNA or an RNA, or itmay contain a mixture of ribonucleotides and deoxyribonucleotides. Mostgenerally the invention provides oligonucleotides or polynucleotidesthat may range in length anywhere from 15 nucleotides to as long as 200nucleotides. The polynucleotides include a first nucleotide sequencethat targets an ICT-1053 gene, or an ICT-1052 gene, or an ICT-1027 gene,or an ICT-1051 gene, or an ICT-1054 gene, or an ICT-1020 gene, or anICT-1021 gene, or an ICT-1022 gene. The first nucleotide sequenceconsists of either a) a targeting sequence whose length is any number ofnucleotides from 15 to 30, or b) a complement thereof. Such apolynucleotide is termed a linear polynucleotide herein.

FIG. 1 provides schematic representations of certain embodiments of thepolynucleotides of the invention. The invention discloses sequences thattarget an ICT-1053 gene, or an ICT-1052 gene, or an ICT-1027 gene, or anICT-1051 gene, or an ICT-1054 gene, or an ICT-1020, gene, or an ICT-1021gene, or an ICT-1022 gene, or in certain cases siRNA sequences that areslightly mismatched from such a target sequence, all of which areprovided in SEQ ID NOS:7-76, 81-84, and 89-242, which are disclosed inExample 1. The sequences disclosed therein range in length from 19nucleotides to 25 nucleotides. The targeting sequences are representedschematically by the lightly shaded blocks in FIG. 1. FIG. 1, Panel A,a) illustrates an embodiment in which the disclosed sequence shown as“SEQ” may optionally be included in a larger polynucleotide whoseoverall length may range up to 200 nucleotides.

The invention additionally provides that, in the targetingpolynucleotide, a sequence chosen from SEQ ID NOS:7-76, 81-84, and89-242 may be part of a longer targeting sequence such that thetargeting polynucleotide targets a sequence in a target gene that islonger than the first nucleotide sequence represented by SEQ. This isillustrated in FIG. 1, Panel A, b), in which the complete targetingsequence is shown by the horizontal line above the polynucleotide, andby the darker shading surrounding the SEQ block. As in all embodimentsof the polynucleotides, this longer sequence may optionally be includedin a still larger polynucleotide of length 200 or fewer bases (FIG. 1,Panel A, b)).

The invention further provides a targeting sequence that is a fragmentof any of the above targeting sequences such that the fragment targets asequence given in SEQ ID NOS:7-76, 81-84, and 89-242 that is at least 15nucleotides in length (and at most 1 base shorter than the reference SEQID NO: illustrated in FIG. 1, Panel A, c)), as well as a targetingsequence wherein up to 5 nucleotides may differ from being complementaryto the target sequence given in SEQ ID NOS:7-76, 81-84, and 89-242(illustrated in FIG. 1, Panel A, d), showing, in this example, threevariant bases represented by the three darker vertical bars).

Still further the invention provides a sequence that is a complement toany of the above-described sequences (shown in FIG. 1, Panel A, e), anddesignated as “COMPL”). Any of these sequences are included in theoligonucleotides or polynucleotides of the invention. Any linearpolynucleotide of the invention may be constituted of only the sequencesdescribed in a)-e) above, or optionally may include additional bases upto the limit of 200 nucleotides. Since RNA interference requires doublestranded. RNAs, the targeting polynucleotide itself may be doublestranded, including a second strand complementary to at least thesequence given by SEQ ID NOS:7-76, 81-84, and 89-242 and hybridizedthereto, or intracellular processes may be relied upon to generate acomplementary strand.

Thus a polynucleotide of the invention most generally may be singlestranded, or it may be double stranded. In still further embodiments,the polynucleotide contains only deoxyribonucleotides, or it containsonly ribonucleotides, or it contains both deoxyribonucleotides andribonucleotides. In important embodiments of the polynucleotidesdescribed herein the target sequence consists of a sequence that may beeither 15 nucleotides (nt), or 16 nt, or 17 nt, or 18 nt, or 19 nt, or20 nt, or 21 nt, or 22 nt, or 23 nt, or 24 nt, or 25 nt, or 26 nt, or 27nt, or 28 nt, or 29, or 30 nt in length. In still additionaladvantageous embodiments the targeting sequence may differ by up to 5bases from complementarity to a target sequence in the viral pathogengenome.

In several embodiments of the invention, the polynucleotide is an siRNAconsisting of the targeting sequence with optional inclusion of a 3′overhang as described herein that may be 1 nt, or 2 nt, or 3 nt, or 4 ntin length; in many embodiments a 3′ overhang is a dinucleotide.

Alternatively, in recognition of the need for a double stranded RNA inRNA interference, the oligonucleotide or polynucleotide may be preparedto form an intramolecular hairpin looped double stranded molecule. Sucha molecule is formed of a first sequence described in any of theembodiments of the preceding paragraphs followed by a short loopsequence, which is then followed in turn by a second sequence that iscomplementary to the first sequence. Such a structure forms the desiredintramolecular hairpin. Furthermore, this polynucleotide is disclosed asalso having a maximum length of 200 nucleotides, such that the threerequired structures enumerated may be constituted in any oligonucleotideor polynucleotide having any overall length of up to 200 nucleotides. Ahairpin loop polynucleotide is illustrated in FIG. 1, Panel B.

The term “complexed DNA” include a DNA molecule complexed or combinedwith another molecule, for example, a carbohydrate, for example, asugar, that a sugar-DNA complex is formed. Such complex, for example, asugar complexed DNA can enhance or support efficient gene delivery viareceptor, for example, glucose can be complexed with DNA and deliveredto a cell via receptor, such as mannose receptor.

“Encapsulated nucleic acids”, including encapsulated DNA or encapsulatedRNA, refer to nucleic acid molecules in microsphere or microparticle andcoated with materials that are relatively non-immunogenic and subject toselective enzymatic degradation, for example, synthesized microspheresor microparticles by the complex coacervation of materials, for example,gelatin and chondroitin sulfate (see, for example, U.S. Pat. No.6,410,517). Encapsulated nucleic acids in a microsphere or amicroparticle are encapsulated in such a way that it retains its abilityto induce expression of its coding sequence (see, for example, U.S. Pat.No. 6,406,719).

Pharmaceutical Compositions Comprising Targeting Polynucleotides

Pharmaceutical compositions for therapeutic applications include one ormore, targeting polynucleotides and a carrier. The pharmaceuticalcomposition comprising the one or more targeting polynucleotide isuseful for treating a disease or disorder associated with the expressionor activity of a Target Gene. Carriers include, but are not limited to,saline, buffered saline, dextrose, water, glycerol, ethanol, andcombinations thereof. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to, pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives.

In many embodiments, the invention relates to a pharmaceuticalcomposition comprising at least two targeting polynucleotides, designedto target different Target Genes, and a pharmaceutically acceptablecarrier. Due of the targeting of mRNA of more than one Target Gene,pharmaceutical compositions comprising a plurality of targetingpolynucleotides may provide improved efficiency of treatment as comparedto compositions comprising a single targeting polynucleotide, at leastin tumor cells expressing these multiple genes. In this embodiment, theindividual targeting polynucleotides are prepared as described in thepreceding section, which is incorporated by reference herein. Onetargeting polynucleotide can have a nucleotide sequence which issubstantially complementary to at least part of one Target Gene;additional targeting polynucleotides are prepared, each of which has anucleotide sequence that is substantially complementary to part of adifferent Target Gene. The multiple targeting polynucleotides may becombined in the same pharmaceutical composition, or formulatedseparately. If formulated individually, the compositions containing theseparate targeting polynucleotides may comprise the same or differentcarriers, and may be administered using the same or different routes ofadministration. Moreover, the pharmaceutical compositions comprising theindividual targeting polynucleotides may be administered substantiallysimultaneously, sequentially, or at preset intervals throughout the dayor treatment period.

The pharmaceutical compositions of the present invention areadministered in dosages sufficient to inhibit expression of the targetgene. The targeting polynucleotides are highly efficient in producing aninhibitory effect, as it is understood that, as part of a RISC complex,they act in a catalytic fashion. Thus compositions comprising the one ormore targeting polynucleotides of the invention can be administered atsurprisingly low dosages.

A maximum dosage of 5 mg targeting polynucleotide per kilogram bodyweight of recipient per day is sufficient to inhibit or completelysuppress expression of the target gene. In general, a suitable dose oftargeting polynucleotide will be in the range of 0.01 to 5.0 milligramsper kilogram body weight of the recipient per day, preferably in therange of 0.1 to 200 micrograms per kilogram body weight (mcg/kg) perday, more preferably in the range of 0.1 to 100 mcg/kg per day, evenmore preferably in the range of 1.0 to 50 mcg/kg per day, and mostpreferably in the range of 1.0 to 25 mcg/kg per day. The pharmaceuticalcomposition may be administered once daily, or the targetingpolynucleotide may be administered as two, three, four, five, six ormore sub-doses at appropriate intervals throughout the day. In thatcase, the targeting polynucleotide contained in each sub-dose must becorrespondingly smaller in order to achieve the total daily dosage. Thedosage unit can also be compounded as a sustained release formulationfor delivery over several days, e.g., using a conventional formulationwhich provides sustained release of the targeting polynucleotide over aseveral day period. Sustained release formulations are well known in theart. In this embodiment, the dosage unit contains a correspondingmultiple of the daily dose.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual targeting polynucleotidesencompassed by the invention can be made using conventionalmethodologies or on the basis of in vivo testing using an appropriateanimal model, and can be adjusted during treatment according toestablished criteria for determining appropriate dose-responsecharacteristics.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases. For example, mouse models areavailable for hematopoietic malignancies such as leukemias, lymphomasand acute myelogenous leukemia. The MMHCC (Mouse models of Human CancerConsortium) web page (emice.nci.nih.gov), sponsored by the NationalCancer Institute, provides disease-site-specific compendium of knowncancer models, and has links to the searchable Cancer Models Database(cancermodels.nci.nih.gov), as well as the NCI-MMHCC mouse repository.Examples of the genetic tools that are currently available for themodeling of leukemia and lymphomas in mice, and which are useful inpracticing the present invention, are described in the followingreferences: Maru, Y., Int. J. Hematol. (2001) 73:308-322; Pandolfi, P.P., Oncogene (2001) 20:5726-5735; Pollock, J. L., et al., Curr. Opin.Hematol. (2001). delta: 206-211; Rego, E. M., et al., Semin. in Hemat.(2001) 38:4-70; Shannon, K. M., et al. (2001) Modeling myeloid leukemiatumors suppressor gene inactivation in the mouse, Semin. Cancer Biol.11, 191-200; Van Etten, R. A, (2001) Curr. Opin. Hematol. 8, 224-230;Wong, S., et al. (2001) Oncogene 20, 5644-5659; Phillips J A., CancerRes. (2000) 52(2): 437-43; Harris, A. W., et al, J. Exp. Med. (1988)167(2): 353-71; Zeng X X et al., Blood. (1988) 92(10): 3529-36;Eriksson, B., et al., Exp. Hematol. (1999) 27(4): 682-8; and Kovalchuk,A., et al., J. Exp. Med. (2000) 192(8): 1183-90. Mouse repositories canalso be found at: The Jackson Laboratory, Charles River Laboratories,Taconic, Harlan, Mutant Mouse Regional Resource Centers (MMRRC) NationalNetwork and at the European Mouse Mutant Archive. Such models may beused for in vivo testing of targeting polynucleotide, as well as fordetermining a therapeutically effective dose. Furthermore variousknock-out or knock-in transgenic animal models for effects of the TargetGenes can be prepared and studied to evaluate dosing of targetingpolynucleotides.

The pharmaceutical compositions encompassed by the invention may beadministered by any means known in the art including, but not limited tooral or parenteral routes, including intravenous, intramuscular,intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal,vaginal and topical (including buccal and sublingual) administration. Incertain embodiments, the pharmaceutical compositions are administered byintravenous or intraparenteral infusion or injection, and in additionalcommon embodiments the pharmaceutical composition comprising targetingpolynucleotides may be delivered directly in situ to a tumor, a canceror a precancerous growth using laparoscopic and similar microsurgicalprocedures.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,the pharmaceutical compositions of the invention will generally beprovided in sterile aqueous solutions or suspensions, buffered to anappropriate pH and isotonicity. Suitable aqueous vehicles includeRinger's solution and isotonic sodium chloride. In a preferredembodiment, the carrier consists exclusively of an aqueous buffer. Inthis context, “exclusively” means no auxiliary agents or encapsulatingsubstances are present which might affect or mediate uptake of targetingpolynucleotide in the cells that express the target gene. Suchsubstances include, for example, micellar structures, such as liposomesor capsids, as described below. Surprisingly, the present inventors havediscovered that compositions containing only naked targetingpolynucleotide and a physiologically, acceptable solvent are taken up bycells, where the targeting polynucleotide effectively inhibitsexpression of the target gene. Although microinjection, lipofection,viruses, viroids, capsids, capsoids, or other auxiliary agents arerequired to introduce targeting polynucleotide into cell cultures,surprisingly these methods and agents are not necessary for uptake oftargeting polynucleotide in vivo. Aqueous suspensions according to theinvention may include suspending agents such as cellulose derivatives,sodium alginate, polyvinyl-pyrrolidone and gum tragacanth, and a wettingagent such as lecithin. Suitable preservatives for aqueous suspensionsinclude ethyl and n-propyl p-hydroxybenzoate.

The pharmaceutical compositions useful according to the invention alsoinclude encapsulated formulations to protect the targetingpolynucleotide against rapid elimination from the body, such as acontrolled release formulation, including implants and microencapsulateddelivery systems. Biodegradable, biocompatible polymers can be used,such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid,collagen, polyorthoesters, and polylactic acid. Methods for preparationof such formulations will be apparent to those skilled in the art. Thematerials can also be obtained commercially from Alza Corporation andNova Pharmaceuticals, Inc. Liposomal suspensions (including liposomestargeted to infected cells with monoclonal antibodies to viral antigens)can also be used as pharmaceutically acceptable carriers. These can beprepared according to methods known to those skilled in the art, forexample, as described in U.S. Pat. No. 4,522,811; PCT publication WO91/06309; and European patent publication EP-A-43075, which areincorporated by reference herein.

In certain embodiments, the encapsulated formulation comprises a viralcoat protein. In this embodiment, the targeting polynucleotide may bebound to, associated with, or enclosed by at least one viral coatprotein. The viral coat protein may be derived from or associated with avirus, such as a polyoma virus, or it may be partially or entirelyartificial. For example, the coat protein may be a Virus Protein 1and/or Virus Protein 2 of the polyoma virus, or a derivative thereof.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds which exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulation a range of dosage for use in humans. The dosage ofcompositions of the invention lies preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays andanimal models to achieve a circulating plasma concentration range of thecompound that includes the IC50 (i.e., the concentration of the testcompound which achieves a half-maximal inhibition of symptoms) asdetermined in cell culture. Such information can be used to moreaccurately determine useful doses in humans.

In addition to their administration individually or as a plurality, asdiscussed above, the targeting polynucleotides useful according to theinvention can be administered in combination with other known agentseffective in treatment of diseases. In any event, the administeringphysician can adjust the amount and timing of targeting polynucleotideadministration on the basis of results observed using standard measuresof efficacy known in the art or described herein.

It is further envisioned to use Intradigm Corporation's proprietary genedelivery technologies for high throughput delivery into animal models.Intradigm's PolyTran™ technology (see International Application No. WO0147496) enables direct administration of plasmids into tumor andachieves a seven-fold increase of efficiency over the gold standardnucleotide delivery reagents. This provides strong tumor expression andactivity of candidate target proteins in the tumor.

Methods for Treating Diseases Caused by Expression of a Target Gene

In certain embodiments, the invention relates to methods for treating asubject having a disease or at risk of developing a disease caused bythe expression of a Target Gene. The one or more targetingpolynucleotides can act as novel therapeutic agents for controlling oneor more of cellular proliferative and/or differentiative disordersincluding a tumor, a cancer, or a precancerous growth. The methodcomprises administering a pharmaceutical composition of targetingpolynucleotides to the patient (e.g., human), such that expression ofthe target gene is silenced. Because of their high specificity, thetargeting polynucleotides of the present invention specifically targetmRNAs of target genes of diseased cells and tissues, as described below,and at surprisingly low dosages.

In the prevention of disease, the target gene may be one which isrequired for initiation or maintenance of the disease, or which has beenidentified as being associated with a higher risk of contracting thedisease. In the treatment of disease, the targeting polynucleotide canbe brought into contact with the cells or tissue exhibiting the disease.For example, targeting polynucleotide comprising a sequencesubstantially complementary to all or part of an mRNA formed in thetranscription of a mutated gene associated with cancer, or one expressedat high levels in tumor cells may be brought into contact with orintroduced into a cancerous cell or tumor.

Examples of cellular proliferative and/or differentiative disordersinclude cancer, e.g., carcinoma, sarcoma, metastatic disorders orhematopoietic neoplastic disorders, e.g., leukemias. A metastatic tumorcan arise from a multitude of primary tumor types, including but notlimited to those of pancreas, prostate, colon, lung, breast and liverorigin. As used herein, the terms “cancer,” “hyperproliferative,” and“neoplastic” refer to cells having the capacity for autonomous growth,i.e., an abnormal state of condition characterized by rapidlyproliferating cell growth. These terms are meant to include all types ofcancerous growths or oncogenic processes, metastatic tissues ormalignantly transformed cells, tissues, or organs, irrespective ofhistopathologic type or stage of invasiveness. Proliferative disordersalso include hematopoietic neoplastic disorders, including diseasesinvolving hyperplastic/neoplastic cells of hematopoietic origin, e.g.,arising from myeloid, lymphoid or erythroid lineages, or precursor cellsthereof.

Combinations of siRNA

Several embodiments of the invention provide pharmaceutical compositionscontaining two or more oligonucleotides or polynucleotides each of whichincludes a sequence targeting genes in the genome of a respiratoryvirus. Related embodiments provide methods of treating cells, andmethods of treating respiratory viral infections, using thecombinations, as well as uses of such combination compositions in themanufacture of pharmaceutical compositions intended to treat respiratoryviral infections. The individual polynucleotide components of thecombination may target different portions of the same gene, or differentgenes, or several portions of one gene as well as more than one gene, inthe genome of the viral pathogen. An advantage of using a combination ofoligonucleotides or polynucleotides is that the benefits of inhibitingexpression of a given gene are multiplied in the combination. Greaterefficacy is achieved in knocking down a gene or silencing a viral genomeby use of multiple targeting sequences. Enhanced efficiency ininhibiting viral replication is achieved by targeting more than one genein the viral genome.

Pharmaceutical Compositions

The targeting polynucleotides of the invention are designated “activecompounds” or “therapeutics” herein. These therapeutics can beincorporated into pharmaceutical compositions suitable foradministration to a subject.

As used herein, “pharmaceutically acceptable carrier” is intended toinclude any and all solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Suitable carriersare described in textbooks such as Remington's Pharmaceutical Sciences,Gennaro AR (Ed.) 20^(th) edition (2000) Williams & Wilkins Pa., USA, andWilson and Gisvold's Textbook of Organic Medicinal and PharmaceuticalChemistry, by Delgado and Remers, Lippincott-Raven, which areincorporated herein by reference. Preferred examples of components thatmay be used in such carriers or diluents include, but are not limitedto, water, saline, phosphate salts, carboxylate salts, amino acidsolutions, Ringer's solutions, dextrose (a synonym for glucose)solution, and 5% human serum albumin. By way of nonlimiting example,dextrose may used as 5% or 10% aqueous solutions. Liposomes andnon-aqueous vehicles such as fixed oils may also be used. The use ofsuch media and agents for pharmaceutically active substances is wellknown in the art. Supplementary active compounds can also beincorporated into the compositions.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral, nasal, inhalation, transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intravenous, intradermal, or subcutaneous applicationcan include the following components: a sterile diluent such as waterfor injection, saline solution, fixed oils, polyethylene glycols,glycerin, propylene glycol or other synthetic solvents; antibacterialagents such as benzyl alcohol or methyl parabens; antioxidants such asascorbic acid or sodium bisulfite; chelating agents such asethylenediaminetetraacetic acid; buffers such as acetates, citrates orphosphates, and agents for the adjustment of tonicity such as sodiumchloride or dextrose.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g., films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releasepharmaceutical active agents over shorter time periods. Advantageouspolymers are biodegradable, or biocompatible. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811. Sustained-release preparations having advantageous forms,such as microspheres, can be prepared from materials such as thosedescribed above.

The siRNA polynucleotides of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by any of a number of routes, e.g., as described in U.S.Pat. Nos. 5,703,055. Delivery can thus also include, e.g., intravenousinjection, local administration (see U.S. Pat. No. 5,328,470) orstereotactic injection (see e.g.; Chen et al. (1994) Proc. Natl. Acad.Sci. USA 91:3054-3057). The pharmaceutical preparation of the genetherapy vector can include the gene therapy vector in an acceptablediluent, or can comprise a slow release matrix in which the genedelivery vehicle is imbedded. Alternatively, where the complete genedelivery vector can be produced intact from recombinant cells, e.g.,retroviral vectors, the pharmaceutical preparation can include one ormore cells that produce the gene delivery system.

The pharmaceutical compositions can be included in a kit, e.g., in acontainer, pack, or dispenser together with instructions foradministration.

Also within the invention is the use of a therapeutic in the manufactureof a pharmaceutical composition or medicament for treating a respiratoryviral infection in a subject.

Delivery

In several embodiments the siRNA polynucleotides of the invention aredelivered by liposome-mediated transfection, for example by usingcommercially available reagents or techniques, e.g., Oligofectamine™,LipofectAmine™ reagent, LipofectAmine 2000™ (Invitrogen), as well as byelectroporation, and similar techniques. Additionally siRNApolynucleotides are, is delivered to animal models, such as rodents ornon-human primates, through inhalation and instillation into therespiratory tract. Additional routes for use with animal models includeintravenous (IV), subcutaneous (SC), and related routes ofadministration. The pharmaceutical compositions containing the siRNAsinclude additional components that protect the stability of siRNA,prolong siRNA lifetime, potentiate siRNA function, or target siRNA tospecific tissues/cells. These include a variety of biodegradablepolymers, cationic polymers (such as polyethyleneimine), cationiccopolypeptides such as histidine-lysine (HK) polypeptides see, forexample, PCT publications WO 01/47496 to Mixson et al., WO 02/096941 toBiomerieux, and WO 99/42091 to Massachusetts Institute of Technology),PEGylated cationic polypeptides, and ligand-incorporated polymers, etc.positively charged polypeptides, PolyTran polymers (naturalpolysaccharides, also known as scleroglucan), a nano-particle consistsof conjugated polymers with targeting ligand (TargeTran variants),surfactants (Infasurf; Forest Laboratories, Inc.; ONY Inc.), andcationic polymers (such as polyethyleneimine). Infasurf® (calfactant) isa natural lung surfactant isolated from calf lung for use inintratracheal instillation; it contains phospholipids, neutral lipids,and hydrophobic surfactant-associated proteins B and C. The polymers caneither be uni-dimensional or multi-dimensional, and also could bemicroparticles or nanoparticles with diameters less than 20 microns,between 20 and 100 microns, or above 100 micron. The said polymers couldcarry ligand molecules specific for receptors or molecules of specialtissues or cells, thus be used for targeted delivery of siRNAs. ThesiRNA polynucleotides are also delivered by cationic liposome basedcarriers, such as DOTAP, DOTAP/Cholesterol (Qbiogene, Inc.) and othertypes of lipid aqueous solutions. In addition, low percentage (5-10%)glucose aqueous solution, and Infasurf are effective carriers for airwaydelivery of siRNA³⁰.

Using fluorescence-labeled siRNA suspended in an oral-tracheal deliverysolution of 5% glucose and Infasurf examined by fluorescence microscopy,it has been shown that after siRNA is delivered to mice via the nostrilor via the oral-tracheal route, and washing the lung tissues the siRNAis widely distributed in the lung (see co-owned WO 2005/01940,incorporated by reference herein in its entirety). The delivery of siRNAinto the nasal passage and lung (upper and deeper respiratory, tract) ofmice was shown to successfully silence the indicator genes (GFP orluciferase) delivered simultaneously with the siRNA in a plasmidharboring a fusion of the indicator gene and the siRNA target (seeco-owned WO 2005/01940). In addition, experiments reported by theinventors, working with others, have demonstrated that siRNA speciesinhibit the replication of SARS coronavirus, thus relieving the lungpathology, in the SARS-infected rhesus monkeys³⁰.

siRNA Recombinant Vectors

Another aspect of the invention pertains to vectors, preferablyexpression vectors, containing an siRNA polynucleotide of the invention.As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “expressionvectors”. In general, expression vectors of utility in recombinant DNAtechniques are often in the form of plasmids. In the presentspecification, “plasmid” and “vector” can be used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors, such asviral vectors (e.g., replication defective retroviruses, adenovirusesand adeno-associated viruses), which serve equivalent functions.

The recombinant expression vectors of the invention comprise a nucleicacid of the invention, in a form suitable for expression of the nucleicacid in a host cell, which means that the recombinant expression vectorsinclude one or more regulatory sequences, selected on the basis of thehost cells to be used for expression, that is operatively linked to thenucleic acid sequence to be expressed. Within a recombinant expressionvector, “operably linked” is intended to mean that the nucleotidesequence of interest is linked to the regulatory sequence(s) in a mannerthat allows for expression of the nucleotide sequence (e.g., in an invitro transcription/translation system or in a host cell when the vectoris introduced into the host cell). The term “regulatory sequence” isintended to includes promoters, enhancers and other expression controlelements (e.g., polyadenylation signals). Such regulatory sequences aredescribed, for example, in Goeddel (1990) GENE EXPRESSION TECHNOLOGY:METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. Regulatorysequences include those that direct constitutive expression of anucleotide sequence in many types of host cell and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). In yet another embodiment, anucleic acid of the invention is expressed in mammalian cells using amammalian expression vector. Examples of mammalian expression vectorsinclude pCDM8 (Seed (1987) Nature 329:840) and pMT2PC (Kaufman et al.(1987) EMBO J6: 187-195). When used in mammalian cells, the expressionvector's control functions are often provided by viral regulatoryelements. For example, commonly used promoters are derived from polyoma,Adenovirus 2, cytomegalovirus and Simian Virus 40. Additional vectorsinclude minichromosomes such as bacterial artificial chromosomes, yeastartificial chromosomes, or mammalian artificial chromosomes. For othersuitable expression systems for both prokaryotic and eukaryotic cells.See, e.g., Chapters 16 and 17 of Sambrook et al., MOLECULAR CLONING: ALABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type such as a cell of the respiratory tract.Tissue-specific regulatory elements are known in the art. The inventionfurther provides a recombinant expression vector comprising a DNAmolecule of the invention cloned into the expression vector. The DNAmolecule is operatively linked to a regulatory sequence in a manner thatallows for expression (by transcription of the DNA molecule) of an RNAmolecule that includes an siRNA targeting a viral RNA. Regulatorysequences operatively linked to a nucleic acid can be chosen that directthe continuous expression of the RNA molecule in a variety of celltypes, for instance viral promoters and/or enhancers, or regulatorysequences can be chosen that direct constitutive, tissue specific orcell type specific expression of antisense RNA.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (2001), Ausubelet al. (2002), and other laboratory manuals.

Method of Treatment

The present invention relates to a method for treating a disease in amammal associated with pathological expression of an ICT-1053 gene, oran ICT-1052 gene, or an ICT-1027 gene, or an ICT-1051 gene, or anICT-1054 gene, or an ICT-1020 gene, or an ICT-1021 gene, or an ICT-1022gene. The method includes administering to the mammal inhibitory nucleicacid compositions that interact with at least one of the targets anICT-1053 gene, or an ICT-1052 gene, or an ICT-1027 gene, or an ICT-1051gene, or an ICT-1054 gene, or an ICT-1020 gene, or an ICT-1021 gene, oran ICT-1022 gene at the DNA or RNA level. The nucleic acid compositionis capable of suppressing the expression of the one or more targets anICT-1053 gene, or an ICT-1052 gene, or an ICT-1027 gene, or an ICT-1051gene, or an ICT-1054 gene, or an ICT-1020 gene, or an ICT-1021 gene, oran ICT-1022 gene when introduced into a tissue of the mammal. The methodof treatment is directed in particular to a disease such as a cancer ora precancerous growth in the tissue of the mammal. Frequently the tissueis a breast tissue, a colon tissue, a prostate tissue, a skin tissue, abone tissue, a parotid gland tissue, a pancreatic tissue, a kidneytissue, a uterine cervix tissue, a lymph node tissue, or an ovariantissue. In common cases the inhibitor is a siRNA, an RNAi, a shRNA, anantisense RNA, an antisense DNA, a decoy molecule, a decoy DNA, a doublestranded DNA, a single-stranded DNA, a complexed DNA, an encapsulatedDNA, a viral DNA, a plasmid DNA, a naked RNA, an encapsulated RNA, aviral RNA, a double stranded RNA, a molecule capable of generating RNAinterference, or combinations thereof.

The following Examples illustrate certain embodiments of the presentinvention. They should not be viewed as limiting the scope of theinvention, which is represented in the specification as a whole, and, inthe claims.

EXAMPLES

Novel target genes for application of RNA interference to the treatmentof cancer were identified, and experiments to assess the tumorinhibition properties of siRNAs directed at the targets were carriedout. Specifically, experiments were done by targeting ICT-1053,ICT-1052, ICT-1027, ICT-1051, ICT-1054, ICT-1020, ICT-1021, ICT-1022,and ICT-1022.

Two siRNA target sequences were selected within each gene and verifiedby BLAST, and the sequences synthesized by Qiagen Inc (Germantown, Md.).In the experiments reported in these Examples, a mixture of two specificsiRNA sequences for each gene was repeatedly delivered to xenograftmodels or to cells in culture. Human VEGF siRNA was used as a positivecontrol against which to assess the effects of the chosen siRNAs.

Example 1 Small Interfering RNA (siRNA)

siRNAs duplexes were made based upon selected targeted regions of theDNA sequences for targets ICT-1052, ICT-1053 or ICT-1027 (SEQ ID NOS:1,3, and 5), or ICT-1051, ICT-1054, ICT-1020, ICT-1021, ICT-1022, orICT-1022. In certain embodiments a designed sequence includesAA-(N)_(m)-TT (where 15≦m≦21) and has a G-C content of about 30% to 70%.If no suitable sequences are found, the fragment size is extended tosequences of up to 29 nucleotides. In certain embodiments the 3′ end ofa polynucleotide has an overhang (i.e. having unpaired bases) given byTT or UU. Without wishing to be bound by theory, it is believed thatsymmetric 3′ overhangs on an siRNA duplex help to ensure that the smallinterfering ribonucleoprotein particles (siRNPs) are formed withapproximately equal ratios of sense and antisense target RNA-cleavingsiRNPs (Elbashir et al. Genes & Dev. 15:188-200, 2001).

ICT-1052 siRNA: Sense or antisense siRNAs of 21 bp were identified basedupon targeted regions of SEQ ID NO:1). These are shown in Table 2.

TABLE 2 siRNA targeted sequences identified in ICT-1052) TARGET SEQUENCESEQ ID NO: AACACCCATCCAGAATGTCAT 7 AAGCCAATTTATCAGGAGGTG 8TCAAGAGCATGAACGCATCAA 9 TGTGTGTTGTATGGTCAATAA 10 ACTGAATGGTACTTCGTATGT11 CCTCGCAAGCAATTGGAAACA 12 CTCTGATAGTGCAGAGACTTA 13GAATGATGCTACTCTGATCTA 14 GCAATACAGTCAAAGTTTCAA 15 TTGTGTGTTGTATGGTCAATA16 TTTGTGTGTTGTATGGTCAAT 17 AGGACTACACACTTGTATATA 18AGAGTATTGTAAATGGTGGAT 19 GAATGGTACTTCGTATGTTAA 20 CTGTAAATTGCGATAAGGAAA21 CCAAATATTGCCGTTTCATAA 22 CAAGAGCATGAACGCATCAAT 23GCATGAACGCATCAATAGAAA 24 GAAGAGCTATTACAATCCAAA 25 CATTACATCATCAGGACTTGA26 ACAGGACTACACACTTGTATA 27 CAGGACTACACACTTGTATAT 28

ICT-1053 siRNA: Sense or antisense siRNAs of 21 bp were identified basedupon targeted regions of SEQ ID NO:3). These are shown in Table 3.

TABLE 3 siRNA targeted sequences identified in ICT-1053. TARGET SEQUENCESEQ ID NO: AATCTGTCTGCAGCCCAGAAC 29 AAGCGTGGAAGTTAACTTCAC 30AGTCATGTATCCTGTGTTTAA 31 GGATATAGCTAGTGCAATAAA 32 ACGCCTTAATGTGTCATTATA33 TTAAAGATGGCAAGGCAATAA 34 CCGCTTTCATCAAGGCTGAAA 35TCGTAAGTGCCAACCGACTAA 36 AGCGATATGCTGCAAGATAAT 37 ACTTAATACTTCAGACCTTCA38 CAGTCATGTATCCTGTGTTTA 39 CTTAATACTTCAGACCTTCAA 40CTGATGATGTAGAAGAGTATA 41 TGAACTGCCTTTATCTGTAAA 42 ACGGATTCAGTTCCAGTTTAA43 TTTAAAGATGGCAAGGCAATA 44 TTAATGAGCTAGAACGAGTAA 45TGAAAGCGATATGCTGCAAGA 46 GAAAGCGATATGCTGCAAGAT 47 CTTGAAAGCGATATGCTGCAA48 TCATAATCTCACACTGAAGAT 49 TATTGCCATCTTACACCATAT 50

ICT-1027 siRNA: Sense or antisense siRNAs of 21 bp were identified basedupon targeted regions of SEQ ID NO:5). These are shown in Table 4.

TABLE 4 siRNA targeted sequences identified in ICT-1027. TARGET SEQUENCESEQ ID NO: AATCCCCAGAGCCAAGGCAGA 51 AAGGGGGGACATCCTCAAGGT 52AGTCCTAGCTGACGCCAATAA 53 GGTAGTGATTAACTGTGAATA 54 CTCCAGTTGTAGCAGGTTTCA55 TTCCTGTGTTCTTCGTATATA 56 TCCATCAGTGCATGACGTTTA 57CCTGTGGTGATGTGCCTGTAA 58 GGAACGTCTAAGAGTCAAGAA 59 AGAAGAAATGCTTAGCAAACA60 TAGTCCTAGCTGACGCCAATA 61 TGACGTTTAAGGCCACGTATA 62CATGAAGCCTTGCTGAACTAA 63 GTCTCCAGAAACCAGCAGATA 64 GTTCCTGTGTTCTTCGTATAT65 CATTTGGTAGGTAGTGATTAA 66 GCTCGATGCCTTTGCTGTTTA 67CTGTGGTGATGTGCCTGTAAT 68 GCATTTGGTAGGTAGTGATTA 69 TCAGCCAATTTGTCTCCTACT70 ATATCATGAAGCCTTGCTGAA 71 TACTAAGCCAGGAGGCTTTAA 72

Additional targeted sequences are identified in Tables 5-18.

TABLE 5 19-nt siRNA targeted sequences identified in  ICT-1053.TARGET SEQUENCE SEQ ID NO: GGCAGCTGATGATGTAGAA 113 GCCAGAATTCCAAGACCTA114 CCAGAATTCCAAGACCTAA 115 GGCACGAGCACTTAAACAA 116 GCACGAGCACTTAAACAAA117 GGTTTCTGCAGACAATCAA 118 GCAGACAATCAAGGATATA 119 CCTCCTGAAGGGATCTAAT120 GGATCTAATCCAGGATGTT 121 GGATGTTGAATGGGATTAT 122

TABLE 6 25-nt siRNA targeted sequences identified in ICT-1053.TARGET SEQUENCE SEQ ID NO: CCTTCTTCGTATGGCAGCTGATGAT 123CAGAGCCAGAATTCCAAGACCTAAA 124 CCAGAATTCCAAGACCTAAACGAAA 125GACAATCAAGGATATAGCTAGTGCA 126 GGCAAGGCAATAAATGTGTTCGTAA 127TCGTAAGTGCCAACCGACTAATTCA 128 CCGACTAATTCATCAAACCAACTTA 129TCAGTCCCTCCTGAAGGGATCTAAT 130 GAAGGGATCTAATCCAGGATGTTGA 131GGGATTATTGCCATCTTACACCATA 132

TABLE 7 19-nt siRNA targeted sequences identified in ICT-1052.TARGET SEQUENCE SEQ ID NO: CCGGTTCATCAACTTCTTT 133 GGACCAGTCCTACATTGAT134 GCACAAAGCAAGCCAGATT 135 GCATGTCAACATCGCTCTA 136 GCTGGTGTTGTCTCAATAT137 GGTGTTGTCTCAATATCAA 138 GCAGTGAATTAGTTCGCTA 139 CCAACTACAGAAATGGTTT140 CCATGTGAACGCTACTTAT 141 GCATCAGAACCAGAGGCTT 142

TABLE 8 25-nt siRNA targeted sequences identified in ICT-1052.TARGET SEQUENCE SEQ ID NO: CAAAGCCAATTTATCAGGAGGTGTT 143CAGTCGGAGGTTCACTGCATATTCT 144 CACACAAGAATAATCAGGTTCTGTT 145CGCTCTAATTCAGAGATAATCTGTT 146 CAGCACTGTTATTACTACTTGGGTT 147CCAGTAGCCTGATTGTGCATTTCAA 148 CAGCCTCCTTCTGGGAGACATCATA 149TGGGAGACATCATAGTGCTAGTACT 150 GCAGGAAATATTGAGGGCTTCTTGA 151GCCACTCATTTAGAATTCTAGTGTT 152

TABLE 9 19-nt siRNA targeted sequences identified in ICT-1027.TARGET SEQUENCE SEQ ID NO: CCGGTTCATCAACTTCTTT 153 GGACCAGTCCTACATTGAT154 GCACAAAGCAAGCCAGATT 155 GCATGTCAACATCGCTCTA 156 GCTGGTGTTGTCTCAATAT157 GGTGTTGTCTCAATATCAA 158 GCAGTGAATTAGTTCGCTA 159 CCAACTACAGAAATGGTTT160 CCATGTGAACGCTACTTAT 161 GCATCAGAACCAGAGGCTT 162

TABLE 10 25-nt siRNA targeted sequences identified in ICT-1027.TARGET SEQUENCE SEQ ID NO: CAAAGCCAATTTATCAGGAGGTGTT 163CAGTCGGAGGTTCACTGCATATTCT 164 CACACAAGAATAATCAGGTTCTGTT 165CGCTCTAATTCAGAGATAATCTGTT 166 CAGCACTGTTATTACTACTTGGGTT 167CCAGTAGCCTGATTGTGCATTTCAA 168 CAGCCTCCTTCTGGGAGACATCATA 169TGGGAGACATCATAGTGCTAGTACT 170 GCAGGAAATATTGAGGGCTTCTTGA 171GCCACTCATTTAGAATTCTAGTGTT 172

TABLE 11 19-nt siRNA targeted sequences identified in ICT-1051.TARGET SEQUENCE SEQ ID NO: GGACTCTGTGAGGAAACAA 173 GCTTCCAGTCAGACGTCTA174 CCACCAGCCAATCAATGTT 175 CCAATCAATGTTCGTCTCT 176 TCTCCAATGGCTGGGATTT177 GGGATTTGTGGCAGGGATT 178 GCAGGGATTCCACTCAGAA 179 GCCATTCAAGGACTCCTCT180 TCCTCTCTTTCTTCACCAA 181 TCTCTTTCTTCACCAAGAA 182

TABLE 12 25-nt siRNA targeted sequences identified  in ICT-1051.TARGET SEQUENCE SEQ ID NO: GGCGGACTCTGTGAGGAAACAAGAA 183GGGTTGTGCTCTACGAGCTTATGAC 184 GCTCTACGAGCTTATGACTGGCTCA 185CACAATTGAGCTGCTGCAACGGTCA 186 CCTTGCCCACCAGCCAATCAATGTT 187ACCAGCCAATCAATGTTCGTCTCTG 188 CCATCTCCAATGGCTGGGATTTGTG 189CCGCCATTCAAGGACTCCTCTCTTT 190 GCCATTCAAGGACTCCTCTCTTTCT 191GGACTCCTCTCTTTCTTCACCAAGA 192

TABLE 13 19-nt siRNA targeted sequences identified in ICT-1054.TARGET SEQUENCE SEQ ID NO: GGCCTGAGAGGTCTCTCGT 193 GCCTGAGAGGTCTCTCGTC194 GAGAGGTCTCTCGTCGCTG 195 CCCATGGCCGCCTACTCTT 196 CCATGGCCGCCTACTCTTA197 GCTCTCAGGGAGGTCTGTG 198

TABLE 14 19-nt siRNA targeted sequences identified in ICT-1054.TARGET SEQUENCE SEQ ID NO: GGAGTCGGCCTGAGAGGTCTCTCGT 199GAGTCGGCCTGAGAGGTCTCTCGTC 200 TCGGCCTGAGAGGTCTCTCGTCGCT 201CCTTGGCCCATGGCCGCCTACTCTT 202

TABLE 15 19-nt siRNA targeted sequences identified in ICT-1020.TARGET SEQUENCE SEQ ID NO: GCTCGAAATCTTACGCAAA 203 GCTTATATCAGTAGCAATT204 GCACCCATCTCTAATTATA 205 GCACTAGAATTTAAACCTA 206 GCCGTTATCATTCCAAGAT207 CCACACATCTTCAAGACTT 208 GCACATCAAGGTGCTAATA 209 GCAATTAATGGTCTTTCTT210 GCAGTTATGATTTAGCTAA 211 GCAACCAACTACCTCATAT 212

TABLE 16 25-nt siRNA targeted sequences identified in ICT-1020.TARGET SEQUENCE SEQ ID NO: CCTGAAATTTGTAACTCCTAAAGTA 213GCAGTTGTCTTAAACAGATTGATAA 214 CAACCTGCTTATTGCAACAAGTATT 215CATCAATAGATACTGTGCTAGATTA 216 TCCAGAGTGTTTGAGGGATAGTTAT 217CCTTTACCTGATGAACTCAACTTTA 218 CAGCATACTGTGTTCTACCTCTTAA 219CCAAATGGGAAAGTCTGCAGAATAA 220 CAGCCGCATGGTGGTGTCAATATTT 221CCGCATGGTGGTGTCAATATTTGAT 222

TABLE 17 19-nt siRNA targeted sequences identified in ICT-1021.TARGET SEQUENCE SEQ ID NO: GCAATACCCAATTTCAATT 223 GAATCTTCCAAAGCGCAAA224 TCTTCCAAAGCGCAAAGAA 225 TCCAAAGCGCAAAGAAGTT 226 CCAAAGCGCAAAGAAGTTA227 GCAAAGAAGTTATTTGCCG 228 GAAGTTATTTGCCGAGGAT 229 TCATTCTCCTTCAAGGGAA230 GCTTGGAGTTTGTCATCCT 231 TCATCCTACACCAACCTAA 232

TABLE 18 25-nt siRNA targeted sequences identified in ICT-1021.TARGET SEQUENCE SEQ ID NO: TCTACATTCCAAGGAGAGATTTAAA 233CACCATGAATCTTCCAAAGCGCAAA 234 CGCAAAGAAGTTATTTGCCGAGGAT 235AGAAGTTATTTGCCGAGGATCTGAT 236 ACAATATCATTCTCCTTCAAGGGAA 237CAATATCATTCTCCTTCAAGGGAAT 238 CAAATGTGTTGTTGAAGCTATTTCT 239GCTTGGAGTTTGTCATCCTACACCA 240 AGTTTGTCATCCTACACCAACCTAA 241CATCCTACACCAACCTAATTCAAAT 242

Example 2 Inhibition of Tumor Growth by ICT-1053 siRNA

MDA-MB-435 human breast carcinoma cells (ATCC, Manassas, Va.) weremaintained in RPMI 1640 media (Sigma-Aldrich, St. Louis, Mo.) with 10%fetal bovine serum (FBS) (20 ml for one T-75 flask) at 37° C. and 5%CO₂. 4×10⁵ MDA-MB-435 cells in 50 μl OPTI-MEM (Invitrogen, Carlsbad,Calif.) were injected into fat pads under the nipples of mice on day 0to induce tumors.

On Day 11 and Day 18, the mice were treated with either 10 ug ICT-1053siRNA (5 ug of ICT-1053-siRNA-a mixed with 5 ug of ICT-1053-siRNA-b) ora negative control of 10 ug non-specific siRNA (NC) in 20 ul of PBS.

The ICT-1053 siRNA-a duplex consists of two complementary polynucleotidestrands having the following sequences:

r(UCUGUCUGCAGCCCAGACA)d(TT) (SEQ ID NO: 73) andr(UGUCUGGGCUGCAGACAGA)d(TT). (SEQ ID NO: 74)

The ICT-1053 siRNA-b duplex consists of two complementary polynucleotidestrands having the following the sequences:

r(GCGUGGAAGUUAACUUCAC)d(TT) (SEQ ID NO: 75) andr(GUGAAGUUAACUUCCACGC)d(TT). (SEQ ID NO: 76)

As a positive control, the tumors were treated with two VEGF siRNAinhibitors. The VEGF-siRNA-a duplex consists of two complementarypolynucleotides having the following sequences:

r(UCGAGACCCUGGUGGACAU)d(TT) (SEQ ID NO: 77) andr(AUGUCCACCAGGGUCUCGA)d(TT). (SEQ ID NO: 78)The VEGF-siRNA-b duplex consists of two complementary polynucleotidehaving the following sequences:

r(GGCCAGCACAUAGGAGAGA)d(TT) (SEQ ID NO: 79) andr(UCUCUCCUAUGUGCUGGCC)d(TT). (SEQ ID NO: 80)

As a negative control (NC-siRNA), the tumors were injected with twogreen fluorescent protein (GFP)-siRNA duplexes that have no homologywith any human or mouse gene sequences. The GFP-siRNA-a duplex and theGFP-siRNA-b duplex sequences are given below in Example 5 (SEQ IDNOS:85-88).

The siRNA duplexes were transfected directly into the tumor xenograftsusing electroporation. Tumor size was monitored by measuring the lengthand width using an external caliper before every siRNA delivery andtwice a week after the last siRNA delivery until the end point ofexperiment. Tumor volume is calculated as

Volume=width²×length×0.52.

The results are shown in FIG. 2. The tumor size obtained upon treatmentwith the ICT-1053 siRNA is much smaller than that found with thenonspecific siRNA, and is essentially indistinguishable from the tumorsize obtained with the VEGF siRNA positive control. This shows thatsiRNA targeting ICT-1053, which knocks down PDCD10 expression,powerfully limits the growth of tumors produced by MDA-MB-435xenografts.

Example 3 Inhibition of Tumor Growth by ICT-1052 siRNA

In general, similar experimental procedures were used as described forExample 2. In the present Example control animals were treated with1×PBS only (Vehicle Control in FIG. 3). On Day 11 and Day 18, the micewere treated with either 10 ug ICT-1052 siRNA (5 ug of ICT-1052-siRNA-amixed with 5 ug of ICT-1052-siRNA-b) or 10 ug non-specific siRNA (NC) in20 ul of PBS.

The ICT-1052 siRNA-a duplex consists of two complementary polynucleotidestrands having the following sequences:

r(CACCCAUCCAGAAUGUCAU)d(TT) (SEQ ID NO: 81) andr(AUGACAUUCUGGAUGGGUG)d(TT). (SEQ ID NO: 82)

The ICT-1052 siRNA-b duplex consists of two complementary polynucleotidestrands having the following sequences:

r(GCCAAUUUAUCAGGAGGUG)d(TT) (SEQ ID NO: 83) andr(CACCUCCUGAUAAAUUGGC)d(TT). (SEQ ID NO: 84)

The VEGF-siRNA-a and the VEGF-siRNA-b duplexes used as the positivecontrol are the same as employed in Example 2 (SEQ ID NOS:77-80).

The results are shown in FIG. 3. It is seen that the ICT-1052 siRNA usedin this Example reduces the tumor size in comparison to the negativecontrols, the PBS vehicle and the nonspecific siRNA (NC-siRNA). Thebeneficial effect of ICT-1052 siRNA was not as effective as VEGF si RNA.These results show that the ICT-1052 siRNA used in this Example, whichknocks down c-Met expression, is effective to inhibit tumor growth ofMDA-MB-435 xenografts.

Example 4 Inhibition of Tumor Growth by ICT-1052 siRNA

A549 human lung carcinoma cells (ATCC, Manassas, Va.) were maintained inDMEM media with 10% fetal bovine serum (FBS) at 37° C. and 5% CO₂. AtDay 0, 1×10⁷ A549 cells in 100 ul DMEM medium without serum wereinoculated s.c. into the back flank of anesthetized nude mice. At Day 6,the size of tumor was measured and animals were randomly assigned totreatment groups.

At Day 7, each tumor was transfected intratumorally with either 10 ugICT-1052 siRNA (5 ug of ICT-1052-siRNA-a mixed with 5 ug ofICT-1052-siRNA-b (SEQ ID NOS:81-84); see Example 3) or 10 ugnon-specific siRNA (NC) in 20 ul of PBS using an electroporationenhanced transfection procedure. Four more siRNA deliveries were carriedout at Day 12, Day 16, Day 20, and Day 27. The tumor sizes were measuredbefore each siRNA delivery, and twice a week after the last siRNAdelivery.

The results are shown in FIG. 4. It was observed that the treatment ofA549 tumor with ICT-1052 siRNA in this Example significantly inhibitsthe growth rate of A549 xenografts compared to tumors treated withnon-specific siRNA. These results show that the ICT-1052 siRNA used inthis Example, which knocks down c-Met expression in the tumor,effectively inhibit the growth of A549 lung tumor.

Example 5 Inhibition of Tumor Growth by ICT-1052 siRNA and ICT-1053siRNA

MDA-MB-435 human breast carcinoma cells were maintained in RPMI 1640media with 10% FBS at 37° C. and 5% CO₂. The cells were transfected withthe same ICT-1053 siRNAs used in Example 2 (SEQ ID NOS:73-76), or withthe ICT-1052 siRNA used in Example 3 (SEQ ID NOS:81-84), atconcentrations of 2 ug siRNA/2×10⁶ cells/200 ul DMEM medium or 5 ugsiRNA/2×10⁶ cells/200 ul DMEM medium, using an electroporation mediatedtransfection method. In the control group, the cells were transfectedwith siRNA targeting green fluorescent protein reporter gene (GFP) atthe same concentrations using an electroporation mediated transfectionmethod. The GFP siRNA is a mixture of equal amount of GFP-siRNA-a duplexand GFP-siRNA-b duplex.

The GFP-siRNA-a duplex consists of two complementary polynucleotideswith the following sequences:

r(GCTGACCCTGAAGTTCATC)d(TT) (SEQ ID NO: 85) andr(GAUGAACUUCAGGGUCAGC)d(TT). (SEQ ID NO: 86)

The GFP-siRNA-b duplex consists of two complementary polynucleotideswith following sequences:

r(GCAGCACGACUUCUUCAAG)d(TT) (SEQ ID NO: 87) andr(CUUGAAGAAGUCGUGCUGC)d(TT). (SEQ ID NO: 88)

At 48 hours post transfection, the cell proliferation activity ismeasured using a Cell Proliferation Kit I (MTT-based, where MTT is3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, orthiazolyl blue) (Roche Diagnostics, Indianapolis, Ind.). The medium ineach well is aspirated, then 2 ml of serum-free DMEM is added into eachwell. 200 uL of MTT stock solution (MTT stock solution: 50 mg MTT in 10mL PBS) is added to each well. The plate is incubated at 37° C. in CO₂incubator for 3 hr. During this incubation period, viable cells convertMTT to a water-insoluble formazan dye. The medium in each well isremoved, but not any of the formazan crystals. 2 mL of acidic isopropylalcohol (500 mL isopropyl alcohol+3.5 mL of 6 N HCl) is added to eachwell, and the crystals are completely dissolved for about 10 min. 100 ulfrom each well are transferred into a 96-well plate, and the absorbanceat 570 nm was read with background subtraction at 650 nm using aMicroplate Reader (Model 680, Bio-Rad, Hercules, Calif.).

The results are shown in FIG. 5. It is seen that ICT-1052 siRNA andICT-1053 siRNA provide 25-30% inhibition of growth of the MDA-MB-435cells at both doses applied, whereas the control samples produce only 5%or less inhibition of growth. These data show that the targeting siRNAsemployed in this study are effective to inhibit the growth of humanbreast carcinoma cells in culture.

Example 6 Inhibition of Proliferation of Cancer Cells by ICT-1052 siRNAand ICT-1053 siRNA

HCT116 human colorectal carcinoma cells were maintained in DMEM mediawith 2.5% FBS at 37° C. and 5% CO₂. The HCT116 cells were transfectedwith the same ICT-1053 siRNA as used in Example 2 (SEQ ID NOS:73-76), orwith the ICT-1052 siRNA used in Example 3 (SEQ ID NOS:81-84), at aconcentration of 5 ug siRNA/2×10⁶ cells/200 ul DMEM medium using anelectroporation mediated transfection method. In the control group, thecells were transfected with NC-siRNA at the same concentration.

At 72 hours post transfection, the cell proliferation activity in thetransfected HCT116 cells was measured using a Cell Proliferation Kit I(Roche Diagnostics, Indianapolis, Ind.) as described in Example 5.

The results are shown in FIG. 6. It was observed that treatment ofICT-1052 siRNA or ICT-1053 siRNA resulted in 25-30% inhibition ofproliferation of HCT116 cells, whereas the NC-siRNA treatment resultedin only 8% cell proliferation inhibition, compared to control cells thatreceived mock treatment. These data demonstrate that the ICT-1052 andICT-1053 siRNAs are effective inhibitors of the cell proliferation ofhuman colon carcinoma cells in culture.

Example 7 Inhibition of Proliferation of Lung Carcinoma Cells byICT-1052

A549 human lung carcinoma cells (ATCC, Manassas, Va.) were maintained inDMEM media with 10% fetal bovine serum (FBS) at 37° C. and 5% CO₂. TheA549 cells were transfected with the same ICT-1052 siRNA as used inExample 3 (SEQ ID NOS:81-84) at a concentration of 5 ug siRNA/2×10⁶cells/200 ul DMEM medium, using an electroporation mediated transfectionmethod. In the control group, the A549 cells were transfected withNC-siRNA as described in Example 2 (SEQ ID NOS:85-88). At 72 hours posttransfection, the cell proliferation activity in the transfected cellswas measured, using a Cell Proliferation Kit I (Roche Diagnostics,Indianapolis, Ind.) as described in Example 5.

The results are shown in FIG. 7. It was observed that treatment ofICT-1052 siRNA resulted in an about 25% cell proliferation inhibition ofA549 cells, whereas the NC-siRNA treatment resulted in only a 5% or lesscell proliferation inhibition, compared to control cells that receivedmock treatment. These data demonstrate that the ICT-1052 siRNA caneffectively inhibit the proliferation of human lung carcinoma cells inculture.

Example 8 Inhibition of Tumor Growth by ICT-1027 siRNA

A similar experimental procedure was used as in Examples 2 and 3, withthe modification that the siRNA was administrated at Day 9, Day 14, andDay 20 (indicated with arrows in FIG. 8). The mice were treated witheither 10 ug ICT-1027 siRNA (5 ug of ICT-1027-siRNA-a mixed with 5 ug ofICT-1027-siRNA-b) or 10 ug GFP-siRNA in 20 ul of PBS.

The ICT-1027 siRNA-a duplex consists of two complementary polynucleotidestrands having the following sequences:

r(GGGGGGACAUCCUCAAGGU)d(TT) (SEQ ID NO: 89) andr(ACCUUGAGGAUGUCCCCCC)d(TT). (SEQ ID NO: 90)

The ICT-1027 siRNA-b duplex consists of two complementary polynucleotidestrands having the following sequences:

r(UCCCCAGAGCCAAGGCAGA)d(TT) (SEQ ID NO: 91) andr(UCUGCCUUGGCUCUGGGGA)d(TT). (SEQ ID NO: 92)

GFP siRNA serves as a negative control, and is a mixture of equal amountof GFP-siRNA-a duplex and GFP-siRNA-b duplex (SEQ ID NOS:85-88), asdescribed in Example 2 4. The siRNA duplexes were intratumorallyinjected into the tumor xenograft.

The results are presented in FIG. 8. It is seen that the ICT-1027 siRNAmixture, which knocks down Grb2 expression, significantly inhibits solidtumor growth of the MDA-MB-435 xenograft, compared to the GFP siRNAcontrol.

Example 9 Promotion of Apoptosis of Tumor Cells by ICT-1027 siRNA

MDA-MB-435 human breast carcinoma cells were maintained in RPMI 1640media with 10% FBS at 37° C. and 5% CO₂. The cells were transfected withICT-1027 siRNA using the sequences described in Example 8 (SEQ IDNOS:89-92) at concentrations of 2 ug siRNA/2×10⁶ cells/200 ul DMEMmedium, or 5 ug siRNA/2×10⁶ cells/200 ul DMEM medium, using anelectroporation mediated transfection method. In the control group, thecells did not received any treatment. In the mock group, the cells weretreated with the same electroporation procedure but without siRNA in themedium. At 48 hours post transfection, the apoptosis activity in thecells was measured by quantitative determination of cytoplasmichistone-DNA fragments, which are indicative of apoptosis, using a CellDeath Detection ELISA kit (Roche Diagnostics). The assay is based on aquantitative sandwich-enzyme-immunoassay principle using mousemonoclonal antibodies directed against DNA and histones, respectively,which allows the specific determination of mono- and oligonucleosomes inthe cytoplasmic fraction of cell lysates. Cells in each well are lysedwith lysis buffer provided with the kit. 20 ul cell lysate from eachwell is transferred into streptavidin-coated microtiter plates andincubated with mouse monoclonal antihistone-biotin antibody and mousemonoclonal anti-DNA-peroxidase. Unbound antibodies are washed out. Theamount of nucleosome is determined quantitatively by evaluatingperoxidase activity photometrically with2,2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid; ABTS) assubstrate. The plate is then placed on a plate reader and the absorbanceis measured at 590 nm using a Microplate Reader (Model 680, Bio-Rad,Hercules, Calif.).

The results are shown in FIG. 9. It is seen that, compared to thecontrol and mock samples, ICT-1027 siRNA, which knocks down Grb2 geneexpression; induces significant apoptosis in a dose-dependent fashion.The results suggest that inhibitory RNA directed against ICT-1027inhibits tumor growth of MDA-MB-435 xenografts (Example 8) by inducingapoptosis of tumor cells.

Example 10 Inhibition of Growth of a Breast Cancer Xenografts byICT-1051 siRNA

A similar experimental procedure as described in Example 2 was used inthis Example to validate ICT-1051 (A-Raf) as a target.

On Day 11, and Day 18, the MDA-MB-435 tumor were treated with either 10ug ICT-1051 siRNA (5 ug of ICT-1051-siRNA-a mixed with 5 ug ofICT-1051-siRNA-b) or 10 ug NC-siRNA.

The ICT-1051 siRNA-a duplex consists of two complementary polynucleotidewith following sequences:

r(GAGUUACCUUCCUAAUGCA)d(TT) (SEQ ID NO: 93) andr(UGCAUUAGGAAGGUAACUC)d(TT). (SEQ ID NO: 94)

The ICT-1051 siRNA-b duplex consists of two complementary polynucleotidewith following sequences:

r(GAUUCCCUUGGUAUAUUCA)d(TT) (SEQ ID NO: 95) andr(UGAAUAUACCAAGGGAAUC)d(TT). (SEQ ID NO: 96)

NC-siRNA serves as a negative control, and is a mixture of equal amountof GFP-siRNA-a duplex and GFP-siRNA-b duplex, as described in Example 2.

The results are presented in FIG. 10. The ICT-1051 siRNA mixture, whichknocks down A-Raf expression, slowed down the growth of the MDA-MB-435xenograft, compared to the NC-siRNA treated xenografts.

Example 11 Inhibition of Growth Breast Cancer Xenografts by ICT-1054siRNA

A similar experimental procedure as described in Example 2 was used inthis Example to validate ICT-1054 (PCDP6) as a target for cancertherapy.

On Day 11, and Day 18, the MDA-MB-435 tumor were treated with either 10ug ICT-1054 siRNA (5 ug of ICT-1054-siRNA-a mixed with 5 ug ofICT-1054-siRNA-b) or 10 ug NC-siRNA.

The ICT-1054 siRNA-a duplex consists of two complementary polynucleotidewith following sequences:

r(GACAGGAGUGGAGUGAUAU)d(TT) (SEQ ID NO: 97) andr(AUAUCACUCCACUCCUGUC)d(TT). (SEQ ID NO: 98)

The ICT-1054 siRNA-b duplex consists of two complementary polynucleotidewith following sequences:

r(CUUCAGCGAGUUCACGGGU)d(TT) (SEQ ID NO: 99) andr(ACCCGUGAACUCGCUGAAG)d(TT). (SEQ ID NO: 100)

NC-siRNA serves as a negative control, and is a mixture of equal amountof GFP-siRNA-a duplex and GFP-siRNA-b duplex, as described in Example 2.

The results are presented in FIG. 11. The ICT-1054 siRNA mixture, whichknocks down PCDP6 expression, slowed down the growth of the MDA-MB-435xenograft, compared to the NC-siRNA treated xenografts.

Example 12 Inhibition of Growth of Breast Cancer Xenografts by ICT-1020

A similar experimental procedure as described in Example 2 was used inthis Example to validate ICT-1020 (Dicer) as a target for cancertherapy, with modified schedule for siRNA administration.

In this Example, the MDA-MB-435 tumor xenografts were treated on Day 9and Day 14 with either 10 ug ICT-1020 siRNA (5 ug of ICT-1020-siRNA-amixed with 5 ug of ICT-1020-siRNA-b) or 10 ug NC-siRNA.

The ICT-1020 siRNA-a duplex consists of two complementary polynucleotidewith following sequences:

r(UGGGUCCUUUCUUUGGACU)d(TT) (SEQ ID NO: 101) andr(AGUCCAAAGAAAGGACCCA)d(TT). (SEQ ID NO: 102)

The ICT-1020 siRNA-b duplex consists of two complementary polynucleotidewith following sequences:

r(CUGCUUGAAGCAGCUCUGG)d(TT) (SEQ ID NO: 103) andr(CCAGAGCUGCUUCAAGCAG)d(TT). (SEQ ID NO: 104)

NC-siRNA serves as a negative control, and is a mixture of equal amountof GFP-siRNA-a duplex and GFP-siRNA-b duplex, as described in Example 2.

The results are presented in FIG. 12. The ICT-1020 siRNA treatment,which specifically knocks down Dicer expression within tumor cells,significantly reduced the growth rate of the MDA-MB-435 xenograft,compared to the xenografts treated with the negative control NC-siRNA.

Example 13 Inhibition of Growth of Breast Cancer Xenografts by ICT-1021siRNA

A similar experimental procedure as described in Example 2 was used inthis Example to validate ICT-1021 (MD2 protein) as a target for cancertherapy. On Day 11 and Day 18, the MDA-MB-435 tumors were treated witheither 10 ug ICT-1021 siRNA (5 ug of ICT-1021-siRNA-a mixed with 5 ug ofICT-1021-siRNA-b) or 10 ug NC-siRNA.

The ICT-1021 siRNA-a duplex consists of two complementary polynucleotidewith following sequences:

r(GCUCAGAAGCAGUAUUGGG)d(TT) (SEQ ID NO: 105) andr(CCCAAUACUGCUUCUGAGC)d(TT). (SEQ ID NO: 106)

The ICT-1021 siRNA-b duplex consists of two complementary polynucleotidewith following sequences:

r(UGCAAUACCCAAUUUCAAU)d(TT) (SEQ ID NO: 107) andr(AUUGAAAUUGGGUAUUGCA)d(TT). (SEQ ID NO: 108)

NC-siRNA serves as a negative control, and is a mixture of equal amountof GFP-siRNA-a duplex and GFP-siRNA-b duplex, as described in Example 2.

The results are presented in FIG. 13. The ICT-1021 siRNA treatment,which specifically knocks down MD2 protein expression within tumorcells, reduced the growth rate of the MDA-MB-435 xenograft, compared tothe NC-siRNA treated xenografts.

Example 14 Inhibition of Growth of Breast Cancer Xenografts by ICT-1022siRNA

A similar experimental procedure as described in Example 2 was used inthis Example to validate ICT-1022 (GAGE-2) as a target for cancertherapy. In this Example the MDA-MB-435 xenografts were treated on Day10 and Day 15 with either 10 ug ICT-1022 siRNA (5 ug of ICT-1022-siRNA-amixed with 5 ug of ICT-1022-siRNA-b) or 10 ug NC-siRNA.

The ICT-1022 siRNA-a duplex consists of two complementary polynucleotidewith following sequences:

r(UGAUUGGGCCUAUGCGGCC)d(TT) (SEQ ID NO: 109) andr(GGCCGCAUAGGCCCAAUCA)d(TT). (SEQ ID NO: 110)

The ICT-1022 siRNA-b duplex consists of two complementary polynucleotidewith following sequences:

r(GUGGAACCAGCAACACCUG)d(TT) (SEQ ID NO: 111) andr(CAGGUGUUGCUGGUUCCAC)d(TT). (SEQ ID NO: 112)

NC-siRNA serves as a negative control, and is a mixture of equal amountof GFP siRNA-a duplex and GFP-siRNA-b duplex, as described in Example 2.

The growth curves of the siRNA treated MDA-MB-435 xenografts arepresented in FIG. 14. The ICT-1022 siRNA treatment, which specificallyknocks down GAGE-2 expression within tumor cells, significantly reducedthe growth rate of the MDA-MB-435 xenograft, compared to the NC-siRNAtreated xenografts.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All references and materials citedherein, including all U.S. and foreign patents and patent applications,are specifically and entirely hereby incorporated herein by reference.It is intended that the specification and examples be consideredexemplary only, with the true scope and spirit of the inventionindicated by the following claims.

REFERENCES

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1. An isolated targeting polynucleotide whose length is 200 or fewernucleotides, the polynucleotide comprising a first nucleotide sequencewherein the first nucleotide sequence targets an ICT-1053 gene, or anICT-1052 gene, or an ICT-1027 gene, or an ICT-1051 gene, or an ICT-1054gene, or an ICT-1020 gene, or an ICT-1021 gene, or an ICT-1022 gene,wherein any T (thymidine) or any U (uridine) may optionally besubstituted by the other and wherein the first nucleotide sequenceconsists of a) a sequence whose length is any number of nucleotides from15 to 30, or b) a complement of a sequence given in a).
 2. (canceled) 3.The polynucleotide according to claim 1 wherein the first nucleotidesequence consists of a) a sequence that targets a sequence chosen fromSEQ ID NOS: 7-76, 81-84, and 89-242; b) an extended sequence longerthan, and comprising, the targeting sequence given in item a), whereinthe extended sequence targets an ICT-1053 gene, or an ICT-1052 gene, oran ICT-1027 gene, or an ICT-1051 gene, or an ICT-1054 gene, or anICT-1020 gene, or an ICT-1021 gene, or an ICT-1022 gene, and thetargeting sequence targets a sequence chosen from SEQ ID NOS: 7-76,81-84, and 89-242; c) a fragment of a sequence that targets a sequencechosen from SEQ ID NOS:7-76, 81-84, and 89-242 wherein the fragmentconsists of a sequence of contiguous bases at least 15 nucleotides inlength and at most one base shorter than the chosen sequence; d) atargeting sequence wherein up to 5 nucleotides differ from a sequencethat targets a sequence chosen from SEQ ID NOS:7-76, 81-84, and 89-242;or e) a complement of a sequence given in a)-d).
 4. The polynucleotideaccording to claim 1 wherein the length of the first nucleotide sequenceis any number of nucleotides from 21 to
 25. 5. The polynucleotideaccording to claim 1 consisting of a sequence chosen from SEQ IDNOS:7-76, 81-84, and 89-242, optionally including a dinucleotideoverhang bound to the 3′ of the chosen sequence. 5a-9. (canceled)
 10. Adouble stranded polynucleotide comprising a first targetingpolynucleotide strand according to claim 1 and a second polynucleotidestrand comprising a second nucleotide sequence that is substantiallycomplementary to at least the first nucleotide sequence of the firstpolynucleotide strand and is hybridized thereto. 11-16. (canceled)
 17. Apharmaceutical composition comprising the polynucleotide according toclaim 1 and a pharmaceutically acceptable carrier. 18-20. (canceled) 21.A method of inhibiting the growth of a cancer cell in a subject,comprising the step of administering to the subject the pharmaceuticalcomposition according to claim
 17. 22. A method of promoting apoptosisin a cancer cell in a subject, comprising the step of administering tothe subject the pharmaceutical composition according to claim
 17. 23-31.(canceled)
 32. A method for decreasing the expression of an ICT-1053gene, an ICT-1052 gene, an ICT-1027 gene, an ICT-1051 gene, an ICT-1054gene, an ICT-1020 gene, an ICT-1021 gene or an ICT-1022 gene in a cell,comprising introducing into the cell the nucleic acid molecule accordingto claim
 1. 33. The polynucleotide according to claim 1, comprising atleast one nucleotide that is modified.
 34. The polynucleotide accordingto claim 33, wherein the at least one modified nucleotide comprises amodification in the phosphate group, the monosaccharide or the base. 35.The polynucleotide according to claim 33, wherein the at least onemodified nucleotide is a nucleotide comprising a 2′-O-methyl ribose. 36.The polynucleotide according to claim 10, wherein the polynucleotide isblunt-ended and wherein the first and the second nucleotide strands areeach 25 nucleotides in length.
 37. The composition according to claim17, further comprising one or more additional targeting polynucleotidesthat induce RNA interference and decrease the expression of a gene ofinterest.
 38. The composition according to claim 17, further comprisinga cationic copolypeptide.
 39. The composition according to claim 38,wherein the cationic copolypeptide is a histidine-lysine copolypeptide.40. The composition according to claim 17, further comprisingpolyethylene glycol.
 41. The composition according to claim 17, furthercomprising a targeting ligand.
 42. An antibody directed against anICT-1053 gene, an ICT-1052 gene, an ICT-1027 gene, an ICT-1051 gene, anICT-1054 gene, an ICT-1020 gene, an ICT-1021 gene or an ICT-1022 geneproduct polypeptide.
 43. A method of treating a cancer, a tumor or aprecancerous growth in a subject, comprising the step of administeringto the subject the antibody according to claim 42.